U.S. patent application number 14/163192 was filed with the patent office on 2014-07-17 for methods of treating complications and disorders associated with g-csf administration.
The applicant listed for this patent is Robert SACKSTEIN. Invention is credited to Robert SACKSTEIN.
Application Number | 20140199316 14/163192 |
Document ID | / |
Family ID | 47108078 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140199316 |
Kind Code |
A1 |
SACKSTEIN; Robert |
July 17, 2014 |
METHODS OF TREATING COMPLICATIONS AND DISORDERS ASSOCIATED WITH
G-CSF ADMINISTRATION
Abstract
The present embodiments relate to novel uses of MPO inhibitors
and inhibitors of MPO and E-selectin binding. In some embodiments,
methods are provided for treating G-CSF-induced vascular
complications and associate tissue injury comprising administering
to a subject in need thereof a compound that inhibits E-selectin
receptor/ligand interaction or inhibits MPO activity. The
inhibitors may be administered in conjunction with G-CSF therapy.
The inhibitors include antibody molecules, as well as homologues,
analogues and modified or derived forms thereof, including
immunoglobulin fragments like Fab, F(ab').sub.2 and Fv, small
molecules, including peptides, oligonucleotides, peptidomimetics
(including aptamers) and organic compounds (e.g.,
glycomimetics).
Inventors: |
SACKSTEIN; Robert; (Sudbury,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SACKSTEIN; Robert |
Sudbury |
MA |
US |
|
|
Family ID: |
47108078 |
Appl. No.: |
14/163192 |
Filed: |
January 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13465691 |
May 7, 2012 |
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14163192 |
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61482784 |
May 5, 2011 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
C07K 16/40 20130101;
A61K 39/3955 20130101; A61K 2039/505 20130101; A61P 9/00
20180101 |
Class at
Publication: |
424/139.1 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. A method for preventing and/or treating G-CSF-induced vascular
complications and associated tissue damage comprising administering
to a subject in need thereof a compound that inhibits the
interaction between E-selectin receptor and MPO-EL, wherein the
compound is administered in conjunction with G-CSF therapy.
2. The method of claim 1, wherein the compound is an anti-MPO-EL
antibody.
3. The method of claim 1, wherein the compound is administered
prior to, during or after administration of G-CSF.
4. A method for preventing and/or treating vascular complications
and associated tissue damage comprising administering to a subject
in need thereof a compound that inhibits the interaction between
E-selectin receptor and MPO-EL.
5. The method of claim 5, wherein the compound that inhibits the
interaction between E-selectin receptor and MPO-EL is an
anti-MPO-EL antibody.
6. The method of claim 4, wherein the vascular complication is
sepsis.
7. The method of claim 4, wherein the vascular complication is
leukocytoclastic vasculitis.
8. The method of claim 4, wherein the vascular complication is
stroke.
9. The method of claim 4, wherein the vascular complication is
angina pectoris.
10. The method of claim 4, wherein the vascular complication is
myocardial infarct.
11. The method of claim 4, wherein the vascular complication is a
localized or systemic vasculitis syndromes.
12. The method of claim 4, wherein the vascular complication is
atherosclerosis.
13. The method of claim 4, wherein the vascular complication is
Wegener's granulomatosis.
14. The method of claim 4, wherein the vascular complication is
sickle cell crises.
15. A method of treating acute myocardial infarction or other
ischemic events in conjunction with reperfusion therapy comprising
administering to a subject in need thereof a compound that inhibits
the interaction between E-selectin receptor and MPO-EL.
16. The method of claim 15, wherein the compound is an anti-MPO-EL
antibody.
17. The method of claim 15, wherein the reperfusion therapy is
primary angioplasty.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 61/482,784, filed May 5, 2011,
incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] Embodiments of the present disclosure are directed to
compositions and methods of treating complications and disorders
associated with G-CSF administration.
BACKGROUND OF THE DISCLOSURE
[0003] The host defense response is critically linked to induction
of vascular adhesion molecules and chemoattractants which recruit
leukocytes to pertinent inflammatory sites. Leukocyte migration to
tissues is tightly regulated in order to ensure optimal delivery of
microbicidal products. This process begins with tethering and
rolling of leukocytes on the endothelial lining at the target
tissue, followed by activation of integrins and firm adhesion to
the vessel wall, culminating in transendothelial migration. The
initial shear-resistant adherence of leukocytes to the endothelial
surface is mediated by selectin receptor/ligand interactions. The
selectin family consists of "leukocyte-specific" L-selectin and
"vascular selectins" P- and E-selectin, each of which binds
sialofucosylated determinants, prototypically displayed as sialyl
Lewis x (sLex). On human hematopoietic cells, two glycoproteins
decorated with sialofucosylated glycans and recognized by mAb
HECA-452 have been characterized as major counter-receptors for the
vascular selectins: a glycoform of P-selectin glycoprotein ligand-1
(PSGL-1) called Cutaneous Lymphocyte Antigen (CLA) and a glycoform
of CD44 known as Hematopoietic Cell E-/L-selectin Ligand
(HCELL).
[0004] Activated leukocytes entering an inflammatory site employ
various cytotoxic mechanisms including generation of reactive
oxygen species (ROS). Phagocytosis induces a respiratory burst
accompanied by creation of superoxide anion (O.sub.2.sup.-) and
hydrogen peroxide (H.sub.2O.sub.2). The lysosomal enzyme
myeloperoxidase (MPO) then uses hydrogen peroxide together with
halide electron donors (Cl.sup.-, I.sup.-) to synthesize toxic and
more efficient ROS like hypohalous acids (HClO, HIO). It is thought
that extracellular leakage/release of toxic oxidants damages
surrounding tissue, including endothelium, resulting in vascular
inflammatory conditions such as leukocytoclastic vasculitis,
systemic vasculitis syndromes, and atherosclerosis.
[0005] Granulocyte colony-stimulating factor (G-CSF or GCSF) is a
hematopoietic cytokine that stimulates leukocyte production and
activation. G-CSF serves a key role in host defense, and its
expression is markedly upregulated in response to inflammatory
insults. G-CSF is commonly used therapeutically to stimulate
myelopoiesis after chemo- and/or radiotherapy and to mobilize
progenitor cells for hematopoietic stem cell transplantation
(HSCT). Though generally safe, use of this cytokine can be
associated with significant vascular complications, including
angina pectoris and myocardial infarct, sickle cell vaso-occlusion,
splenic rupture, and leukocytoclastic vasculitis. Indeed, cutaneous
leukocytoclastic vasculitis has been observed in as many as 6% of
patients receiving G-CSF, and G-CSF administration is known to
induce flares of systemic vasculitis and localized vasculitis
(e.g., uveitis).
[0006] Following G-CSF administration for mobilization of
hematopoietic stem cells, circulating myeloid cells exhibit
increased adhesive interactions with cytokine-stimulated vascular
endothelium when compared to native leukocytes (NL). These
G-CSF-mobilized leukocytes (ML) display increased E-selectin ligand
activity resulting from G-CSF-induced expression of Golgi
glycosyltransferases which control synthesis of sLex.
Conspicuously, G-CSF induces expression of a novel E-selectin
ligand, a glycoprotein with electrophoretic mobility of .about.65
kDa.
[0007] There is increasing evidence of vascular complications,
including angina pectoris, myocardial infarct, and early restenosis
of vascular stents, associated with clinical G-CSF administration.
G-CSF has gained wide therapeutic use for hastening recovery of
neutropenia induced by radiotherapy and/or chemotherapy, in
treatment of cyclic neutropenia, and for mobilization of bone
marrow progenitors for hematopoietic stem cell transplantation.
Moreover, this agent is being considered for treatment of
non-hematologic indications, including immunomodulation and
neuroprotection. Accordingly, there remains a need to critically
examine and prevent the negative effect(s) of G-CSF administration
on vascular/tissue integrity, while retaining intended salutary
effect(s). More generally, there is a need to identify molecular
effectors of vascular/tissue injury that accompany release of
myeloid cells from the marrow, and to therapeutically prevent these
vasculopathic and organopathic effects.
[0008] Throughout this description, including the foregoing
description of related art, any and all publicly available
documents described herein, including any and all U.S. patents, are
specifically incorporated by reference herein in their entirety.
The foregoing description of related art is not intended in any way
as an admission that any of the documents described therein,
including pending United States patent applications, are prior art
to embodiments of the present disclosure. Moreover, the description
herein of any disadvantages associated with the described products,
methods, and/or apparatus, is not intended to limit the disclosed
embodiments. Indeed, embodiments of the present disclosure may
include certain features of the described products, methods, and/or
apparatus without suffering from their described disadvantages.
SUMMARY OF THE DISCLOSURE
[0009] The present application describes a novel composition of the
lysosomal enzyme, MPO. The present application also provides
description of G-CSF-dependent toxicity that is mediated by
induction of MPO expression. Furthermore, the embodiments relate to
novel uses of MPO inhibitors and inhibitors of MPO and E-selectin
binding. The inhibitors include antibody molecules, as well as
homologues, analogues and modified or derived forms thereof,
including immunoglobulin fragments like Fab, F(ab').sub.2 and Fv,
small molecules, including peptides, oligonucleotides,
peptidomimetics (including aptamers) and organic compounds (e.g.,
glycomimetics).
[0010] Embodiments of the present disclosure are directed to
compositions of matter and to methods of treating complications and
disorders associated with leukocyte expression of a
sialofucosylated glycoform of myeloperoxidase (MPO) that serves as
a potent E-selectin ligand. This molecule is known as
MPO-E-selectin Ligand (MPO-EL). MPO-EL may be present on myeloid
cells because of a genetic propensity. Most commonly, MPO-EL is
induced on myeloid cells by G-CSF administration and is also
expressed on myeloid cells in leukemoid reactions (i.e., reactive
leukocytosis).
[0011] According to some embodiments, methods are provided for
treating vascular complications arising from myeloid cell MPO-EL
expression comprising administering to a subject in need thereof a
compound that inhibits the interaction between E-selectin receptor
and an MPO (such as MPO-EL), wherein the compound is an anti-MPO-EL
antibody; in treatment of G-CSF-induced vascular complications,
this compound is administered in conjunction with G-CSF therapy. In
some embodiments, the compound is administered prior to, during or
after administration of G-CSF. In some embodiments, the compound is
given in absence of exogenous G-CSF administration, such as in
cases of leukemoid reactions or certain leukemias (e.g., acute
myelogenous leukemia M3). In some embodiments, the subject is a
human. In some embodiments, the MPO or MPO-EL is human MPO or human
MPO-EL.
[0012] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications comprising
administering to a subject in need thereof an inhibitor of
myeloperoxidase enzymatic activity, wherein the inhibitor is
administered in conjunction with G-CSF therapy. In some
embodiments, the compound is administered prior to, during or after
administration of G-CSF.
[0013] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications comprising
administering to a subject in need thereof a compound that inhibits
the interaction between E-selectin receptor and an MPO (such as
MPO-EL), wherein the compound is an anti-MPO-EL antibody, and
wherein the compound is administered in conjunction with G-CSF
therapy. In some embodiments, the compound is administered prior
to, during or after administration of G-CSF. In some embodiments,
the subject is a human. In some embodiments, the MPO or MPO-EL is
human MPO or human MPO-EL.
[0014] According to some embodiments, methods are provided for
preventing and/or treating G-CSF-induced vascular complications
comprising administering to a subject in need thereof an inhibitor
of myeloperoxidase enzymatic activity, wherein the inhibitor is
administered in conjunction with G-CSF therapy. In some
embodiments, the compound is administered prior to, during or after
administration of G-CSF.
[0015] According to some embodiments, methods are provided for
treating vascular complications comprising administering to a
subject in need thereof a compound that inhibits the interaction
between E-selectin receptor and MPO-EL. In some embodiments, the
compound that inhibits the interaction between E-selectin receptor
and MPO-EL is an anti-MPO-EL antibody. In some embodiments, the
subject is a human. In some embodiments, the MPO-EL is human
MPO-EL.
[0016] According to some embodiments, methods are provided for
treating vascular complications comprising administering to a
subject in need thereof an inhibitor of MPO-EL enzymatic activity.
In some embodiments, the subject is a human. In some embodiments,
the MPO-EL is human MPO-EL.
[0017] According to some embodiments, methods are provided for
preventing and/or treating G-CSF-induced inflammatory complications
comprising administering to a subject in need thereof an inhibitor
of myeloperoxidase enzymatic activity, wherein the inhibitor is
administered in conjunction with G-CSF therapy. In some
embodiments, the compound is administered prior to, during or after
administration of G-CSF.
[0018] According to some embodiments, methods are provided for
preventing and/or treating G-CSF-induced inflammatory complications
comprising administering to a subject in need thereof a compound
that inhibits the interaction between E-selectin receptor and
MPO-EL. In some embodiments, the compound that inhibits the
interaction between E-selectin receptor and MPO-EL is an
anti-MPO-EL antibody. In some embodiments, the subject is a human.
In some embodiments, the MPO-EL is human MPO-EL.
[0019] According to some embodiments, methods are provided for
preventing and/or treating G-CSF-induced inflammatory complications
comprising administering to a subject in need thereof an inhibitor
of MPO-EL enzymatic activity. In some embodiments, the subject is a
human. In some embodiments, the MPO-EL is human MPO-EL.
[0020] In some embodiments, the complication is sepsis.
[0021] In some embodiments, the complication is leukocytoclastic
vasculitis.
[0022] In some embodiments, the complication is angina
pectoris.
[0023] In some embodiments, the complication is myocardial
infarct.
[0024] In some embodiments, the complication is systemic vasculitis
syndromes.
[0025] In some embodiments, the complication is localized
vasculitis syndromes.
[0026] In some embodiments, the complication is stroke.
[0027] In some embodiments, the complication is
atherosclerosis.
[0028] In some embodiments, the complication is Wegener's
granulomatosis.
[0029] In some embodiments, the complication is sickle cell
crises.
[0030] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications and associated tissue
injury comprising administering to a subject in need thereof an
inhibitor of myeloperoxidase enzymatic activity, wherein the
inhibitor is administered in conjunction with G-CSF therapy. In
some embodiments, the compound is administered prior to, during or
after administration of G-CSF.
[0031] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications and associated tissue
injury comprising administering to a subject in need thereof a
compound that inhibits the interaction between E-selectin receptor
and an MPO (such as MPO-EL), wherein the compound is an anti-MPO-EL
antibody, and wherein the compound is administered in conjunction
with G-CSF therapy. In some embodiments, the compound is
administered prior to, during or after administration of G-CSF.
[0032] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications and associated tissue
injury comprising administering to a subject in need thereof an
inhibitor of myeloperoxidase enzymatic activity, wherein the
inhibitor is administered in conjunction with G-CSF therapy. In
some embodiments, the compound is administered prior to, during or
after administration of G-CSF.
[0033] According to some embodiments, methods are provided for
treating vascular complications and associated tissue injury
comprising administering to a subject in need thereof a compound
that inhibits the interaction between E-selectin receptor and
MPO-EL. In some embodiments, the compound that inhibits the
interaction between E-selectin receptor and MPO-EL is an
anti-MPO-EL antibody.
[0034] According to some embodiments, methods are provided for
treating vascular complications and associated tissue injury
comprising administering to a subject in need thereof an inhibitor
of MPO-EL enzymatic activity. According to some embodiments, a
method is provided for treating myocardial infarction comprising
administering to a subject in need thereof an inhibitor of MPO-EL
enzymatic activity.
[0035] According to some embodiments, a method is provided for
treating myocardial infarction and myocardial ischemia comprising
administering to a subject in need thereof a compound that inhibits
the interaction between E-selectin receptor and MPO-EL. In some
embodiments, the compound is an anti-MPO-EL antibody.
[0036] According to some embodiments, a method is provided for
preventing the occurrence of restenosis at a vascular site of a
subject in need thereof comprising administering to a subject in
need thereof an inhibitor of MPO-EL enzymatic activity.
[0037] According to some embodiments, a method is provided for
preventing the occurrence of restenosis at a vascular site of a
subject in need thereof comprising administering to a subject in
need thereof a compound that inhibits the interaction between
E-selectin receptor and MPO-EL. In some embodiments, the compound
is an anti-MPO-EL antibody.
[0038] According to some embodiments, a method is provided for
treating acute myocardial infarction or other ischemic events in
conjunction with reperfusion therapy comprising administering to a
subject in need thereof a compound that inhibits the interaction
between E-selectin receptor and MPO-EL. In some embodiments, the
compound is an anti-MPO-EL antibody.
[0039] According to some embodiments, a method is provided for
treating acute myocardial infarction or other ischemic events in
conjunction with reperfusion therapy comprising administering to a
subject in need thereof an inhibitor of MPO-EL enzymatic
activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1 A-C. G-CSF-mobilized leukocytes depress cardiac
function in mouse myocardial infarct model and exhibit exaggerated
angiotoxicity which is mediated by E-selectin receptor/ligand
interactions. (A) Relative change in the heart ejection fraction
(EF) of induced-myocardial infarct mice injected with PBS, NL or ML
with respect to the heart ejection fraction of sham-operated
counterpart mice injected with PBS, NL or ML. Values represent
mean.+-.SD of percent EF change (n.gtoreq.3) registered after 3
days or 7 days post myocardial infarct, *p<0.05. (B) Endothelial
cell death evaluated in TNF.alpha.-stimulated HUVEC monolayers in
the absence of input leukocytes (No L) or in the presence of NL or
ML. (C) Endothelial cell death in TNF.alpha.-stimulated HUVEC
monolayers was monitored after 48 h incubation with NL, BM cells,
and ML, or with growth media alone (No L; control), in the absence
(-) or presence (+) of function-blocking anti-E-selectin
antibodies. Values represent mean.+-.SD of percent endothelial cell
death (n=10 donors of NL, BM and ML; brackets show statistically
significant differences, * p<0.05).
[0041] FIGS. 2 A-C. Identification of the .about.65 kDa E-selectin
ligand. (A) Representative results of lysates of ML (Lane 1) and
WGA lectin chromatography-purified glycoproteins (Lane 2) resolved
in parallel on reducing 7.5% SDS-PAGE gel and immunoblotted with
E-selectin-Ig chimera. As shown in Lane 1, E-selectin ligands are
present at .about.140 kDa (PSGL-1), .about.100 kDa (HCELL) and at
.about.65 kDa, each of which are preserved and concentrated after
lectin chromatography (Lane 2). (B) In-gel trypsin digestion and
mass spectrometry (MS) analysis of .about.65 kDa lectin-purified
glycoprotein. MPO (MS profile shown) was identified as the main
protein. (C) Representative results of western blots of MPO
immunoprecipitates (IP) from ML resolved under non-reducing (Lane
1) or reducing conditions (Lanes 2 and 3) and stained with anti-MPO
mAb. In non-reduced gels, the mature homodimer of .about.140 kDa is
evident (Lane 1). Under reducing conditions, western blot with
anti-MPO mAb 2C7 (Lane 2) reveals bands at .about.90 kDa
(precursor) and .about.65 kDa (heavy chain), or only the .about.65
kDa band when stained with anti-MPO mAb 3D3 (Lane 3).
[0042] FIGS. 3 A-D. Catalytically active MPO is expressed on the
cell surface of G-CSF mobilized leukocytes. Membrane expression of
MPO was monitored in subsets of native or G-CSF mobilized
leukocytes. (A) Representative MPO flow cytometry histograms are
shown of native granulocytes (NG) and native mononuclear cells (NM)
in comparison to mobilized granulocytes (MG) and mobilized
mononuclear cells (MM). (B) Cumulative results of flow cytometry
analysis for MPO cell surface expression typical for mononuclear
cells and granulocytes. Values represent mean.+-.SD of percent
positive cells from multiple donors (n=15), *p<0.001. (C)
Representative western blot of MPO immunoprecipitates (mAb 2C7) of
lysates of surface biotinylated (+) or non-biotinylated (-) ML and
NL resolved on reducing SDS-PAGE gel. Biotin-labeled (membrane
expressed) MPO was revealed with Streptavidin-HRP. (D) MPO activity
on the surface of NL and ML was evaluated by spectrophotometric
detection of OPD, a chromogenic peroxidase product (n=5 donors of
NL or ML).
[0043] FIGS. 4 A-D. G-CSF induces expression of MPO-EL, an
E-selectin binding glycoform of MPO. Representative blots of
lysates of NL and ML are shown. (A) MPO immunoprecipitates (IPs;
mAb 3D3) resolved on reducing SDS-PAGE and stained with E-selectin
Ig chimera. (B) MPO IPs from lysates of BM, NL and ML stained in
western blot with anti-MPO Ab 3D3. (C) Western blot of membrane in
(B) stripped and probed with E-selectin Ig. (D) MPO IPs from BM, NL
and ML treated (+) or not treated (-) with G-CSF and stained with
anti-MPO mAb 3D3. (E) Western blot of membrane in (D) stripped and
probed with E-selectin Ig.
[0044] FIGS. 5 A-H. N-glycan processing is required for G-CSF
induced surface MPO expression and HECA-452 reactivity. (A)
Representative flow cytometry plots of NL and ML double-stained
with FITC-conjugated HECA-452 and PE-conjugated anti-MPO mAb. (B)
Cumulative flow cytometry results of NL and ML co-stained with
HECA-452 and anti-MPO mAb. Values represent mean.+-.SD of percent
positive cells from multiple donors (n=12), *p<0.001. (C and D)
Flow cytometry analysis of NL, BM and ML cultured for 48 h without
G-CSF (-) or with G-CSF (+): (C) Anti-MPO mAb membrane staining,
values represent mean.+-.SD of percent positive cells from multiple
donors (n>30), *p<0.001; (D) HECA-452 staining, values
represent mean.+-.SD of mean channel fluorescence of cells from
multiple donors (n>30), *p<0.01. (E and F) Flow cytometry
analysis of BM cells treated with neuraminidase and cultured with
G-CSF or G-CSF and DMJ: (E) HECA-452 ligand expression and (F) MPO
expression. (E, F panel 1) BM treated with neuraminidase and
cultured for 48 h (+NA after 48 h) or buffer alone (-NA after 48
h). (E panel 1) Neuraminidase efficiency was confirmed before cell
culturing (+NA after 1 h). (E, F panel 2) BM cells treated with
neuraminidase and cultured for 48 h without G-CSF (+NA after 48 h)
or with G-CSF (+NA+G-CSF after 48 h). (E, F panel 3) BM cells
treated with neuraminidase and cultured for 48 h with G-CSF
(+NA+G-CSF after 48 h) or with GCSF and DMJ (+NA+G-CSF+DMJ after 48
h). (G) Spectrophotometric detection of membrane MPO activity from
NL cultured in the presence or absence of G-CSF and DMJ. Values
represent the relative change in absorbance with respect to
substrate alone (n=5), *p<0.001. (H) Endothelial cell death was
evaluated after co-culture with NL treated with or without G-CSF
and DMJ. Values represent mean.+-.SD of percent endothelial cell
death (n=5), *p<0.05.
[0045] FIGS. 6 A-C. Interruption of E-selectin receptor/ligand
interactions and MPO activity blunts angiotoxicity and myocardial
injury. (A) Percentage endothelial cell death was evaluated in
HUVEC monolayers in the presence (+) or absence (-) of G-CSF,
without (no L) or in co-culture with myeloid cells (NL, BM or ML).
Myeloid cell angiotoxicity was evaluated in the presence (+) or
absence (-) of an E-selectin blocking antibody or of MPO inhibitor
(4-ABAH). Values represent mean.+-.SD of percent endothelial cell
death from multiple donors (n>20), *p<0.05. (B) Evaluation of
angiotoxicity in HUVEC monolayers in the absence or presence of
sialidase or in the presence of ML or sialidase-treated ML. Values
represent mean.+-.SD of percent endothelial cell death from
multiple donors (n=5), *p<0.05. (C) Relative change in the heart
ejection fraction (EF) of induced-myocardial infarct mice injected
with ML or sialidase-treated ML with respect to the heart ejection
fraction of sham-operated counterpart mice injected with ML or
sialidase-treated ML. Values represent mean.+-.SD of percent EF
change (n=3), *p<0.05.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present application describes a composition of matter, a
novel glycoform of MPO that is expressed as a catalytically-active
membrane molecule that binds to E-selectin. This molecule is known
as MPO-E-selectin Ligand (MPO-EL). The application also relates to
the use of inhibitors of E-selectin receptor/ligand interactions
and inhibitors of myeloperoxidase (MPO) and MPO-mediated
cytotoxcity in the treatment and prevention of vascular
inflammatory conditions and related tissue injury. In some
embodiments, the interruption of E-selectin receptor/ligand
interactions or inhibition of myeloperoxidase activity will be of
clinical benefit in ameliorating not only G-CSF-induced vascular
complications, but, also, sepsis, sickle cell crises,
atherosclerosis, and systemic vasculitic syndromes such as
Wegener's granulomatosis.
[0047] According to some embodiments, methods are provided for
treating vascular complications and attendant tissue injury arising
from myeloid cell MPO-EL expression comprising administering to a
subject in need thereof a compound that inhibits the interaction
between E-selectin receptor and an MPO (such as MPO-EL). In some
embodiments, the compound is an anti-MPO-EL antibody. Where the
treatment is related to G-CSF-induced vascular complications, this
compound is administered in conjunction with G-CSF therapy. In some
embodiments, the compound is administered prior to, during or after
administration of G-CSF. In some embodiments, the compound is given
in absence of exogenous G-CSF administration, such as in cases of
leukemoid reactions or certain leukemias (e.g., acute myelogenous
leukemia M3). In some embodiments, the subject is a human. In some
embodiments, the MPO or MPO-EL is human MPO or human MPO-EL.
[0048] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications and attendant tissue
injury by administering to a subject in need thereof inhibitors of
E-selectin receptor/ligand interactions. In some embodiments, the
inhibitors of E-selectin receptor/ligand interactions are
inhibitors of E-selectin receptor/MPO interactions. In some
embodiments, the inhibitors of E-selectin receptor/ligand
interactions are inhibitors of E-selectin receptor/MPO-EL
interactions. In some embodiments, the subject is a human
subject.
[0049] According to some embodiments, methods are provided for
treating G-CSF-induced vascular complications and attendant tissue
injury by administering to a subject inhibitors of MPO enzymatic
activity. In some embodiments, the MPO is human MPO or human
MPO-EL.
[0050] In some embodiments, an inhibitor of the present embodiments
is administered in conjunction with G-CSF therapy. In some
embodiments, the inhibitor is administered to a subject prior to
receiving G-CSF therapy. In some embodiments, the inhibitor is
administered to a subject simultaneously with G-CSF therapy. In
some embodiments, the inhibitor is administered to a subject after
receiving G-CSF therapy. In some embodiments, the inhibitor is
given to a person suffering from sepsis, sickle cell disease, or
collagen vascular disease, such as Wegener's granulomatosis.
[0051] According to some embodiments, methods are provided for
treating vascular complications and attendant tissue injury by
administering to a subject inhibitors of E-selectin receptor/ligand
interactions. In some embodiments, the inhibitors of E-selectin
receptor/ligand interactions are inhibitors of E-selectin
receptor/MPO interactions. In some embodiments, the inhibitors of
E-selectin receptor/ligand interactions are inhibitors of
E-selectin receptor/MPO-EL interactions.
[0052] According to some embodiments, methods are provided for
treating vascular complications and attendant tissue injury by
administering to a subject inhibitors of MPO enzymatic activity. In
some embodiments, the MPO is human MPO or human MPO-EL.
[0053] In another aspect, the invention features a method for
treating a subject who has received, or is scheduled to receive
granulocyte colony stimulating factor (G-CSF). The method includes:
administering to the subject an agent which inhibits
E-selectin-mediated interaction with a selectin ligand. In some
embodiments, the selectin ligand is MPO. In some embodiments, the
selectin ligand is MPO-EL. In various embodiments, the method
reduces side effects due to administration of G-CSF, such as
enhanced leukocyte-endothelial interactions that are associated
with adverse inflammatory reactions.
Methods of Reducing Inflammation
[0054] Inflammation is inhibited (e.g., reduced) by administering
to tissue an inhibitor of the present embodiments (e.g., inhibitors
of E-selectin receptor/MPO interactions or MPO enzymatic activity).
Inhibitors of MPO-EL would affect endothelial beds within tissues
at all sites of inflammation. Tissues that may be treated include
any tissue subject to inflammation such as a gastrointestinal
tissue (e.g., intestinal tissue), a neurologic tissue (e.g.,
brain), cardiac tissue, skeletal tissue, muscular tissue, an
epithelial tissue, an endothelial tissue, a vascular tissue, a
connective tissue, an ocular tissue, a genitourinary tissue (e.g.,
kidney), a pulmonary tissue, a dermal tissue, a lymphatic tissue
(e.g., spleen), or a hepatic tissue. For example, the tissue is an
epithelial tissue such as an intestinal epithelial tissue,
pulmonary epithelial tissue, dermal tissue (i.e., skin), or liver
epithelial tissue.
[0055] Inhibition of inflammation is characterized by a reduction
of redness, pain and swelling of the treated tissue compared to a
tissue that has not been contacted with a selectin inhibitor.
Tissues are directly contacted with an inhibitor. Alternatively,
the inhibitor is administered systemically. The inhibitors of the
present embodiments are administered in an amount sufficient to
decrease (e.g., inhibit) leukocyte-endothelial interaction.
[0056] In some embodiments, the selectin inhibitor is administered
to a subject prior to, during or after receiving G-CSF therapy. An
inflammatory response is evaluated morphologically by observing
tissue damage, localized redness, and swelling of the affected
area. Alternatively, an inflammatory response is evaluated by
measuring c-reactive protein, or IL-1 in the tissue or in the serum
or plasma. Efficaciousness of treatment is determined in
association with any known method for diagnosing or treating the
particular inflammatory disorder. Alleviation of one or more
symptoms of the inflammatory disorder indicates that the compound
confers a clinical benefit.
[0057] The methods described herein lead to a reduction in the
severity or the alleviation of one or more symptoms of an
inflammatory disorder. The inflammatory disorder is acute or
chronic. For example, the methods described herein reduce the
severity of vascular and inflammatory complications associated with
G-CSF therapy. Complications associated with G-CSF therapy include,
for example, respiratory distress syndrome, angina pectoris,
myocardial infarct, cutaneous leukocytoclastic vasculitis,
arthritis, precipitate sickle cell vaso-occlusion, and cardiac
ischemia. Disorders are diagnosed and or monitored, typically by a
physician using standard methodologies.
[0058] The subject is preferably a mammal. The mammal can be, e.g.,
a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
The subject suffers from a disorder in which G-CSF therapy is
indicated. For example, the subject is receiving or is scheduled to
receive a hematopoietic stem cell transplant.
[0059] According to some embodiments, methods are provided for the
prevention of restenosis, said method comprising administering to a
subject in need thereof an inhibitor of MPO, an inhibitor of MPO or
MPO-EL, or an inhibitor of MPO-EL and E-Selectin.
[0060] In some embodiments, methods are provided for at least
slowing the progression of, if not preventing the occurrence of,
restenosis at a vascular site of a host. In the some embodiments,
the target vascular site is treated with an inhibitor of MPO, an
inhibitor of MPO-EL, or an inhibitor of MPO-EL and E-Selectin for a
period of time sufficient for the progression of restenosis at the
target site to at least be slowed.
[0061] The target vascular site that is contacted with the
inhibitors of the present embodiments during the subject methods is
one that has been previously treated for vascular occlusion, where
the occlusion may be a partial or total occlusion. As such, the
target vascular site is one that has the potential for restenosis,
i.e. renarrowing of the vessel walls. The target vessel may be an
artery or vein, and is usually an artery. The vascular site may be
a peripheral or coronary vascular site, where the term peripheral
is used broadly to refer to any site that is not a coronary
vascular site. As such, peripheral vascular sites include not only
limbic vascular sites but also core body vascular sites, e.g.
carotid arteries, renal arteries, etc. In certain embodiments, the
vascular site is a limbic peripheral vascular site, by which is
meant that the vessel in which the vascular site is located is a
vessel found in one of the extremities of the patient to be
treated, i.e. the arms or legs. Often, the vascular site is a site
in a lower extremity vessel, e.g. a lower extremity artery. As
indicated above, of particular interest in certain embodiments are
peripheral arterial vascular sites, where specific peripheral
arteries of interest include: iliac arteries, femoropopliteal
arteries, infrapopliteal arteries, femoral arteries, superficial
femoral arteries, popliteal arteries, and the like. In yet other
embodiments, the vascular site is present in a heart associated
vessel, e.g. the aorta, a coronary artery or branch vessel thereof,
etc. In yet other embodiments, the vascular site is present in a
carotid artery or a branch vessel thereof.
[0062] The vascular site is characterized by having been treated
for vessel narrowing or occlusion prior to practice of the subject
methods. The vessel may have been treated for a total or partial
occlusion, where the nature of the occlusion may vary greatly.
Thus, the vessel may have been subject to an angioplasty or
atherectomy procedure, where the initial vessel narrowing lesion
has been manipulated in some fashion to enhance the blood flow rate
through the vascular site. For example, the vascular site may be
one that has been subjected to balloon angioplasty. Alternatively,
the vascular site may be one that has been subjected to mechanical
removal of at least a portion of the initially present lesion. In
any event, the vascular site is one that is at least potentially
subject to vessel renarrowing or reconstriction. In other words,
the target vascular site is a site that has a propensity for vessel
renarrowing, i.e. restenosis, to occur.
[0063] According to some embodiments, there is provided methods of
using an inhibitor of MPO, an inhibitor of MPO-EL, or an inhibitor
of MPO-EL and E-Selectin in conjunction with reperfusion therapy in
the treatment of acute myocardial infarction or other ischemic
events. This treatment can be used alone or in combination with
other well-known methods of treatment.
[0064] More particularly, the invention provides methods of
treating acute myocardial infarction (AMI) by the administration of
G-CSF polypeptide in conjunction with reperfusion therapy.
[0065] According to some embodiments, there is provided methods for
the treatment of acute myocardial infarction and myocardial
ischemia. In some embodiments, the methods are useful in
conjunction with reperfusion therapy for minimizing tissue damage
and improving patient outcome after such myocardial injury,
illustratively through prevention of cardiac wall thickness loss
ordinarily attending ischemia in affected tissues.
[0066] In one aspect, therefore, methods are provided for treating
AMI to reduce heart damage. Such a method generally would comprise
administering an effective amount of a composition comprising a an
inhibitor of MPO, an inhibitor of MPO-EL, or an inhibitor of MPO-EL
and E-Selectin to a subject in need thereof, including humans,
commencing before, concurrently with, and/or after reperfusion
therapy.
[0067] In another aspect, methods are provided for treating an
ischemic injury. Such a method generally would comprise
administering an effective amount of a composition comprising an
inhibitor of MPO, an inhibitor of MPO-EL, or an inhibitor of MPO-EL
and E-Selectin to a subject in need thereof, commencing before,
concurrently with, or after reperfusion therapy. The reperfusion
therapy contemplated includes mechanical (primary angioplasty),
chemical (administration of a thrombolytic agent), or surgical
(coronary bypass surgery) means.
[0068] In some embodiments, methods are provided for minimizing
tissue damage and improving patient outcome after such myocardial
injury. Such a method generally would comprise administering an
effective amount of a composition comprising an inhibitor of MPO,
an inhibitor of MPO-EL, or an inhibitor of MPO-EL and E-Selectin to
a subject in need thereof.
[0069] In some embodiments, methods are provided for using an
inhibitor of MPO, an inhibitor of MPO-EL, or an inhibitor of MPO-EL
and E-Selectin in conjunction with reperfusion therapy protocols
for the treatment of AMI. The present section provides an overview
of the events which take place in myocardial infarction and
reperfusion to the extent that such a description will facilitate a
better understanding of the methods of the present invention.
[0070] Occlusion of the left coronary artery due to thrombosis is
the major cause of AMI accompanied by ST-segment elevation. The
loss of blood flow to the tissue from the inclusion causes damaged
myocardium due to ischemia, infarction, necrosis, and scar
formation. The expedient restoration of blood flow to the
jeopardized area minimizes tissue damage and improves patient
outcome. This restoration of blood flow, "reperfusion,", can be
accomplished medically, with a thrombolytic agent, or mechanically,
with balloon angioplasty or stenting (Lange et al., N. Engl. J.
Med. 346:954-955, 2002).
[0071] Although reperfusion therapy provides relief to the damaged
tissue and inhibits further scarring of the myocardium, the
infarcted myocardium does not regenerate. Thus, AMI is a critical
event which can lead to progressive heart failure and even death.
Therefore, the present invention provides a novel method of using
an inhibitor of MPO, an inhibitor of MPO-EL, or an inhibitor of
MPO-EL and E-Selectin in conjunction with reperfusion therapy to
minimize myocardial damage after AMI.
Myeloperoxidase
[0072] Myeloperoxidase (MPO) is a highly characterized glycoprotein
well known in the art. MPO is a heme protein synthesized during
myeloid differentiation that constitutes the major component of
neutrophil azurophilic granules. Produced as a single chain
precursor, myeloperoxidase is subsequently cleaved into a light and
heavy chain. The mature myeloperoxidase is a tetramer composed of 2
light chains and 2 heavy chains. This enzyme produces hypohalous
acids central to the microbicidal activity of netrophils.
[0073] The catalytic activity of MPO is located in the heme pocket
deeply embedded in the inner protein core (Fiedler et al., J Biol
Chem. 2000 Apr. 21; 275(16):11964-71.). N-glycosylation sites are
well conserved in the protein, which includes five Asn
glycosylation sites (323, 355, 391, 483, and 729) on the heavy
polypeptide of MPO. MPO is characterized by rigid heme
architecture, as the heme is covalently linked to Asp260, Glu408,
and Met409 and the proximal histidine (His502) is hydrogen-bonded
with an asparagine (Asn587). The roof of the distal heme pocket is
formed by an arginine (Arg405) and the distal histidine (His261)
that is close to calcium-binding Asp262. Additional Ca2+-binding
residues are found in the sequence Thr334Ser335Phe336Va1337Asp338
Ala339Ser340 that is connected to Asn355 by a .alpha.-helix.
Antwerpen et al., J Biol Chem. 2010 May 21; 285(21):
16351-16359.
[0074] The overall protein fold of MPO was first revealed by a 3
.ANG. resolution crystal structure of the canine enzyme (Protein
Data Bank (PDB) code: 1 MYP) (Zeng J, Fenna R E. J Mol Biol. 1992;
226:185-207.). By now, the structure of human MPO at 2.3 .ANG.
resolution is solved and refined to 1.8 .ANG. using X-ray data
recorded at .about.180.degree. C. (1CXP) (Fiedler T J, Davey C A,
Fenna R E. J Biol Chem. 2000; 275:11964-11971). The structure of
MPO and its interaction with bromide (Br--) (1 D2V) and thiocyanate
(SCN--) (1 DNU) (Id., Fiedler et al., 2000) as well as of the
MPO-cyanide (MPO-CN-) complex (1 D5L) and its interaction with Br--
(1 D7W) and SCN-- (1 DNW) was published (Blair-Johnson M, Fiedler
T, Fenna R. Biochemistry. 2001; 40:13990-13997). In addition, there
is one report on the crystal structure of salicylhydroxamic acid
(SHA) bound to human MPO (Davey C A, Fenna R E. Biochemistry. 1996;
35:10967-10973).
[0075] MPO-EL, is a glycoform of a well-characterized lysosomal
enzyme, myeloperoxidase (MPO). MPO-EL is expressed on the myeloid
cell surface and it is catalytically active (i.e., as a
myeloperoxidase). MPO-EL binds E-selectin (an endothelial
molecule), which means that a cytotoxic molecule (MPO) is now
capable of attaching directly to the endothelium. This kills the
endothelial cell, and leads to vascular complications. Inhibitors
(or antagonists) of MPO-EL block the cytotoxic activity by reducing
MPO activity and/or reducing the interaction between MPO-EL and
E-selectin binding. Inhibitors include antibodies to E-selectin, or
small molecules that block E-selectin receptor/ligand interactions
(e.g., GMI-1070)).
MPO Antibodies
[0076] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is an anti-MPO antibody. In some embodiments,
the MPO is human MPO or human MPO-EL.
[0077] In some embodiments, the inhibitor of E-selectin
receptor/MPO interactions suitable for use in the methods of the
present embodiments is an anti-MPO antibody. In some embodiments,
the inhibitor of MPO enzymatic activity suitable for use in the
methods of the present embodiments is an anti-MPO antibody.
[0078] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is an anti-MPO-EL antibody. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is an anti-MPO-EL
antibody.
[0079] The present disclosure provides antibodies that specifically
bind to MPO or MPO-EL. MPO antibodies suitable for use in the
present application are disclosed in U.S. Publication No.
20090148866 and U.S. Publication No. 20080286818, the disclosures
of which are incorporated herein by reference in their
entireties.
[0080] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies (fully or partially humanized),
animal antibodies such as, but not limited to, a bird (for example,
a duck or goose), a shark or whale, a mammal, including a
non-primate (for example, a cow, pig, camel, llama, horse, goat,
rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, etc) or
a non-human primate (for example, a monkey, such as a cynomologous
monkey, a chimpanzee, etc), recombinant antibodies, chimeric
antibodies, single-chain Fvs ("scFv"), single chain antibodies,
single domain antibodies, Fab fragments, F(ab') fragments,
F(ab').sub.2 fragments, disulfide-linked Fvs ("sdFv"), and
anti-idiotypic ("anti-Id") antibodies (including, for example,
anti-Id antibodies to antibodies of the present disclosure), and
functionally active epitope-binding fragments of any of the
above.
[0081] In particular, antibodies include immunoglobulin molecules
and immunologically active fragments of immunoglobulin molecules,
namely, molecules that contain an antigen binding site.
Immunoglobulin molecules can be of any type (for example, IgG, IgE,
IgM, IgD, IgA and IgY), class (for example, IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2) or subclass. An
antibody whose affinity (namely, K.sub.D, k.sub.D or k.sub.a) has
been increased or improved via the screening of a combinatory
antibody library that has been prepared using bio-display, is
referred to herein as an "affinity maturated antibody". For
simplicity sake, an antibody against a protein is frequently
referred to herein as being either an "anti-protein antibody", or
merely a "protein antibody" (e.g., an MPO antibody or an anti-MPO
antibody).
Methods of Making and Using MPO Antibodies
[0082] The antibodies of the present disclosure can be made using a
variety of different techniques known in the art. For example,
polyclonal and monoclonal antibodies against MPO can be raised by
immunizing a suitable subject (such as, but not limited to, a
rabbit, goat, mouse or other mammal) with an immunogenic
preparation which contains a suitable immunogen, such as purified
MPO antigen. For example, a suitable immunogen can be MPO purified
from human neutrophils.
[0083] The antibodies raised in the subject can then be screened to
determine if the antibodies bind to MPO. Such antibodies can be
further screened to identify antibodies that inhibit MPO binding or
interaction with E-selectin. Antibodies can be further screened to
identify antibodies that inhibit MPO activity. Suitable methods to
identify an antibody with the desired characteristics are described
herein (See, Examples section). In some embodiments, the assay
involves assessing whether there was diminished death of
cytokine-activated endothelial cells (e.g., using tumor necrosis
factor alpha (TNF.alpha.) to upregulate endothelial E-selectin
expression) by MPO-EL-expressing myeloid cells (i.e., G-CSF-treated
myeloid cells). Data provided herein shows that inhibition of MPO
function or of E-selectin receptor/ligand interactions blunts
MPO-EL-mediated cytotoxicity. Agents may be screened for their
ability to disrupt MPO-EL-mediated cytotoxicity.
[0084] In some embodiments, there is provided methods of screening
for agents that block MPO-EL enzymatic activity using the methods
disclosed herein.
[0085] In some embodiments, there is provided methods of screening
for agents that block MPO-EL and E-selectin receptor binding
activity using the methods disclosed herein.
[0086] In some embodiments, there is provided methods of screening
for agents that block MPO-EL and E-selectin receptor binding
activity comprising assessing whether there is diminished death of
cytokine-activated endothelial cells (e.g., using tumor necrosis
factor alpha (TNF.alpha.) to upregulate endothelial E-selectin
expression) by MPO-EL-expressing myeloid cells (i.e., G-CSF-treated
myeloid cells).
[0087] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by injection with the native protein, or a
synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example,
recombinantly expressed MPO-EL glycoprotein or a chemically
synthesized MPO polypeptide. The preparation can further include an
adjuvant. Various adjuvants used to increase the immunological
response include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.), human
adjuvants such as Bacille Calmette-Guerin and Corynebacterium
parvum, or similar immunostimulatory agents. If desired, the
antibody molecules directed against HCELL can be isolated from the
mammal (e.g., from the blood) and further purified by well known
techniques, such as protein A chromatography to obtain the IgG
fraction.
[0088] The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope of MPO. A
monoclonal antibody composition thus typically displays a single
binding affinity for a particular MPO glycoprotein with which it
immunoreacts. For preparation of monoclonal antibodies directed
towards a particular MPO glycoprotein, or derivatives, fragments,
analogs or homologs thereof, any technique that provides for the
production of antibody molecules by continuous cell line culture
may be utilized. Such techniques include, but are not limited to,
the hybridoma technique (see Kohler & Milstein, 1975 Nature
256: 495-497); the trioma technique; the human B-cell hybridoma
technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the
EBV hybridoma technique to produce human monoclonal antibodies (see
Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY,
Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be
utilized in the practice of the present invention and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc
Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells
with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96). Each of the above citations are incorporated herein by
reference in their entirety.
[0089] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to a MPO (see
e.g., U.S. Pat. No. 4,946,778). In addition, methodologies can be
adapted for the construction of F.sub.ab expression libraries (see
e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and
effective identification of monoclonal F.sub.ab fragments with the
desired specificity for a MPO or derivatives, fragments, analogs or
homologs thereof. Non-human antibodies can be "humanized" by
techniques well known in the art. See e.g., U.S. Pat. No.
5,225,539. Antibody fragments that contain the idiotypes to a MPO
may be produced by techniques known in the art including, but not
limited to: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv) F,
fragments.
[0090] Additionally, recombinant anti-MPO antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in International Application No. PCT/US86/02269;
European Patent Application No. 184,187; European Patent
Application No. 171,496; European Patent Application No. 173,494;
PCT International Publication No. WO 86/01533; U.S. Pat. No.
4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol.
139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature
314:446-449; Shaw et al. (1988) J Natl Cancer Inst 80:1553-1559);
Morrison (1985) Science 229:1202-1207; Oi et al. (1986)
BioTechniques 4:214; Jones et al. (1986) Nature 321:552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)
J Immunol 141:4053-4060. Each of the above citations are
incorporated herein by reference in their entirety.
[0091] Human monoclonal antibodies can be produced by introducing
an antigen into immune deficient mice that have been engrafted with
human antibody-producing cells or tissues (for example, human bone
marrow cells, peripheral blood lymphocytes (PBL), human fetal lymph
node tissue, or hematopoietic stem cells). Such methods include
raising antibodies in SCID-hu mice (See, for example, WO 93/05796,
U.S. Pat. No. 5,411,749; or McCune et al., Science, 241:1632-1639
(1988)) or Rag-1/Rag-2 deficient mice. Human antibody-immune
deficient mice are also commercially available. For example, Rag-2
deficient mice are available from Taconic Farms (Germantown,
N.Y.).
[0092] In one embodiment, methodologies for the screening of
antibodies that possess the desired specificity include, but are
not limited to, enzyme-linked immunosorbent assay (ELISA) and other
immunologically-mediated techniques known within the art. In a
specific embodiment, selection of antibodies that are specific to a
particular domain of a MPO is facilitated by generation of
hybridomas that bind to the fragment of a MPO possessing such a
domain. Antibodies that are specific for a N-linked glycosylation
site, or derivatives, fragments, analogs or homologs thereof, are
also provided herein.
[0093] In a given embodiment, antibodies for MPO, or derivatives,
fragments, analogs or homologs thereof, that contain the antibody
derived binding domain, are utilized as pharmacologically-active
compounds [hereinafter "Therapeutics"].
Generation of Monoclonal Antibodies (MAbs) to Human MPO
[0094] In one embodiment of the invention, anti-MPO MAbs can be
raised by immunizing rodents (e.g. mice, rats, hamsters and guinea
pigs) with either native MPO purified from human plasma or urine,
or recombinant MPO or its fragments expressed by either eukaryotic
or prokaryotic systems. Other animals can be used for immunization,
e.g. non-human primates, transgenic mice expressing human
immunoglobulins and severe combined immunodeficient (SCID) mice
transplanted with human B lymphocytes. Hybridomas can be generated
by conventional procedures by fusing B lymphocytes from the
immunized animals with myeloma cells (e.g. Sp2/0 and NS0), as
described by G. Kohler and C. Milstein (Nature, 1975: 256:
495-497). In addition, anti-MPO antibodies can be generated by
screening of recombinant single-chain Fv or Fab libraries from
human B lymphocytes in phage-display systems. The specificity of
the MAbs to human MPO can be tested by enzyme linked immunosorbent
assay (ELISA), Western immunoblotting, or other immunochemical
techniques. The inhibitory activity of the antibodies on complement
activation can be assessed by hemolytic assays using unsensitized
rabbit or guinea pig red blood cells (RBCs) for the alternative
pathway, and using sensitized chicken or sheep RBCs for the
classical pathway. The hybridomas in the positive wells are cloned
by limiting dilution. The antibodies are purified for
characterization for specificity to human MPO by the assays
described above.
[0095] If used in treating diseases in humans, the anti-MPO
antibodies would preferably be used as chimeric, deimmunized,
humanized or human antibodies. Such antibodies can reduce
immunogenicity and thus avoid human anti-mouse antibody (HAMA)
response. It is preferable that the antibody be IgG4, IgG2, or
other genetically mutated IgG or IgM which does not augment
antibody-dependent cellular cytotoxicity (S. M. Canfield and S. L.
Morrison, J. Exp. Med., 1991: 173: 1483-1491) and complement
mediated cytolysis (Y. Xu et al., J. Biol. Chem., 1994: 269:
3468-3474; V. L. Pulito et al., J. Immunol., 1996; 156:
2840-2850).
[0096] Chimeric antibodies are produced by recombinant processes
well known in the art, and have an animal variable region and a
human constant region. Humanized antibodies have a greater degree
of human peptide sequences than do chimeric antibodies. In a
humanized antibody, only the complementarity determining regions
(CDRs) which are responsible for antigen binding and specificity
are animal derived and have an amino acid sequence corresponding to
the animal antibody, and substantially all of the remaining
portions of the molecule (except, in some cases, small portions of
the framework regions within the variable region) are human derived
and correspond in amino acid sequence to a human antibody. See L.
Riechmann et al., Nature, 1988; 332: 323-327; G. Winter, U.S. Pat.
No. 5,225,539; C. Queen et al., U.S. Pat. No. 5,530,101.
[0097] Deimmunized antibodies are antibodies in which the T and B
cell epitopes have been eliminated, as described in International
Patent Application PCT/GB98/01473. They have no immunogenicity, or
reduced immunogenicity, when applied in vivo.
[0098] Human antibodies can be made by several different ways,
including by use of human immunoglobulin expression libraries
(Stratagene Corp., La Jolla, Calif.) to produce fragments of human
antibodies (VH, VL, Fv, Fd, Fab, or F(ab').sub.2), and using these
fragments to construct whole human antibodies using techniques
similar to those for producing chimeric antibodies. Human
antibodies can also be produced in transgenic mice with a human
immunoglobulin genome. Such mice are available from Abgenix, Inc.,
Fremont, Calif., and Medarex, Inc., Annandale, N.J.
[0099] One can also create single peptide chain binding molecules
in which the heavy and light chain Fv regions are connected. Single
chain antibodies ("ScFv") and the method of their construction are
described in U.S. Pat. No. 4,946,778. Alternatively, Fab can be
constructed and expressed by similar means (M. J. Evans et al., J.
Immunol. Meth., 1995; 184: 123-138). All of the wholly and
partially human antibodies are less immunogenic than wholly murine
MAbs, and the fragments and single chain antibodies are also less
immunogenic. All these types of antibodies are therefore less
likely to evoke an immune or allergic response. Consequently, they
are better suited for in vivo administration in humans than wholly
animal antibodies, especially when repeated or long-term
administration is necessary. In addition, the smaller size of the
antibody fragment may help improve tissue bioavailability, which
may be critical for better dose accumulation in acute disease
indications.
Inhibitors of myeloperoxidase Derivatives of thioxanthines
[0100] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of thioxanthine.
[0101] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of thioxanthine. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
thioxanthine.
[0102] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of thioxanthine. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
thioxanthine.
[0103] Derivatives of thioxanthine suitable for use in the methods
of the present embodiments are disclosed in U.S. Publication No.
2008/0293748, U.S. Pat. No. 8,026,244, U.S. Pat. No. 7,943,625,
U.S. Pat. No. 7,425,560, each of which are incorporated herein by
reference in their entireties.
[0104] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound according to
following Formula (I):
##STR00001##
[0105] wherein R.sup.1 is selected from C.sub.1-C.sub.6 alkyl, and
said C.sub.1-C.sub.6 alkyl is substituted with C.sub.1-C.sub.6
alkoxy; and at least one of said C.sub.1-C.sub.6 alkyl or said
C.sub.1-C.sub.6 alkoxy is branched; or a pharmaceutically
acceptable salt thereof, solvate or solvate of a salt thereof.
[0106] In some embodiments, the C.sub.1-C.sub.6 alkyl of R.sup.1
represents C.sub.2-4alkyl. In some embodiments, the alkyl is
selected from isobutyl, ethyl and propyl. In some embodiments, the
alkyl is substituted with C.sub.1-3alkoxy. In some embodiments, the
alkyl is substituted with C.sub.1-alkoxy. In some embodiments, the
alkyl is substituted with C.sub.2-alkoxy. In some embodiments, the
alkyl is substituted with propoxy or iso-propoxy.
[0107] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound selected from
the group consisting of:
3-(2-Ethoxy-2-methylpropyl)-2-thioxanthine;
3-(2-Propoxy-2-methylpropyl)-2-thioxanthine;
3-(2-Methoxy-2-methylpropyl)-2-thioxanthine;
3-(2-isopropoxyethyl)-2-thioxanthine;
3-(2-Ethoxypropyl)-2-thioxanthine;
3-(2S-Ethoxypropyl)-2-thioxanthine;
3-(2R-Ethoxypropyl)-2-thioxanthine; or a pharmaceutically
acceptable salt, solvate or solvate of a salt thereof.
[0108] With regard to Formula I, the term "C.sub.1-C.sub.6 alkyl"
referred to herein denotes a straight or branched chain alkyl group
having from 1 to 6 carbon atoms. Examples of such groups include
methyl, ethyl, n-propyl, n-butyl, iso-butyl, tert-butyl, pentyl and
hexyl.
[0109] With regard to Formula I, the term "C.sub.2-C.sub.4 alkyl"
is to be interpreted analogously. It is to be understood that when
the alkyl denotes a C.sub.1 or a C.sub.2 alkyl, such alkyls cannot
be branched.
[0110] With regard to Formula I, the term "C.sub.1-C.sub.6 alkoxy"
referred to herein denotes a straight or branched chain alkoxy
group having from 1 to 6 carbon atoms. Examples of such groups
include methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy,
iso-butoxy, tert-butoxy and pentoxy.
[0111] With regard to Formula I, the term "C.sub.1-C.sub.3 alkoxy"
is to be interpreted analogously. It is to be understood that when
the alkoxy denotes a C.sub.1 or a C.sub.2-alkoxy, such alkoxys
cannot be branched.
[0112] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound according to
following Formulas (Ia) or (Ib):
##STR00002##
[0113] wherein:
[0114] one of X and Y represents S, and the other represents O or
S; R.sup.1 represents hydrogen or C1 to 6 alkyl; R.sup.2 represents
hydrogen or C1 to 6 alkyl; said alkyl group being optionally
substituted by:
[0115] i) a saturated or partially unsaturated 3- to 7-membered
ring optionally incorporating one or two heteroatoms selected
independently from O, N and S, and optionally incorporating a
carbonyl group; said ring being optionally substituted by one or
more substituents selected from halogen, hydroxy, C1 to 6 alkoxy
and C1 to 6 alkyl; said alkyl being optionally further substituted
by hydroxy or C1 to 6 alkoxy; or
[0116] ii) C1 to 6 alkoxy; or
[0117] iii) an aromatic ring selected from phenyl, furyl or
thienyl; said aromatic ring being optionally further substituted by
halogen, C1 to 6 alkyl or C1 to 6 alkoxy; R.sup.3 and R.sup.4
independently represent hydrogen or C1 to 6 alkyl; or a
pharmaceutically acceptable salt thereof.
[0118] The compounds of formulas I, Ia or Ib may exist in
enantiomeric forms. Therefore, all enantiomers, diastereomers,
racemates and mixtures thereof are included within the scope of the
invention.
[0119] It will be appreciated that when R.sup.3 in formulae (Ia)
and (Ib) represents hydrogen, the two alternative representations
(Ia) and (Ib) are tautomeric forms of the same compound. All such
tautomers and mixtures of tautomers are included within the scope
of the present invention.
[0120] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound according to
formula (Ia) or (Ib) wherein at least one of X and Y represents S,
and the other represents O or S; R.sup.1 represents hydrogen or C1
to 6 alkyl; R.sup.2 represents hydrogen or C1 to 6 alkyl; said
alkyl group being optionally substituted by C3 to 7 cycloalkyl, C1
to 4 alkoxy, or an aromatic ring selected from phenyl, furyl or
thienyl; said aromatic ring being optionally further substituted by
halogen, C1 to 4 alkyl or C1 to 4 alkoxy; R.sup.3 and R.sup.4
independently represent hydrogen or C1 to 6 alkyl; or a
pharmaceutically acceptable salt, enantiomer or racemate
thereof.
[0121] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound according to
formula (Ia) or (Ib) wherein at least one of X and Y represents S,
and the other represents O or S; R.sup.1 represents hydrogen or C1
to 6 alkyl; R.sup.2 represents hydrogen or C1 to 6 alkyl; said
alkyl group being optionally substituted by: i) a saturated or
partially unsaturated 3- to 7-membered ring optionally
incorporating one or two heteroatoms selected independently from O,
N and S, and optionally incorporating a carbonyl group; said ring
being optionally substituted by one or more substituents selected
from halogen, hydroxy, C1 to 6 alkoxy and C1 to 6 alkyl; said alkyl
being optionally further substituted by hydroxy or C1 to 4 alkoxy;
or ii) C1 to 4 alkoxy; or iii) an aromatic ring selected from
phenyl, furyl or thienyl; said aromatic ring being optionally
further substituted by halogen, C1 to 4 alkyl or Cl to 4 alkoxy;
R.sup.3 and R.sup.4 independently represent hydrogen or C1 to 6
alkyl.
[0122] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound according to
compounds of formula (Ia) or (Ib) wherein X represents S and Y
represents O.
[0123] In another embodiment, R.sup.3 in formula (Ia) or (Ib)
represents hydrogen.
[0124] In another embodiment, R.sup.2 in formula (Ia) or (Ib)
represents optionally substituted C1 to 6 allyl.
[0125] In another embodiment, R.sup.2 in formula (Ia) or (Ib)
represents C1 to 6 alkyl substituted by a saturated or partially
unsaturated 3- to 7-membered ring optionally incorporating one or
two heteroatoms selected independently from O, N and S, and
optionally incorporating a carbonyl group; said ring being
optionally substituted by one or more substituents selected from
halogen, hydroxy, C1 to 6 alkoxy and C1 to 6 alkyl; said alkyl
being optionally further substituted by hydroxy or C1 to 6
alkoxy.
[0126] In another embodiment, R.sup.2 in formula (Ia) or (Ib)
represents methylene, ethylene or trimethylene substituted by
cyclopropyl, cyclohexyl, tetrahydrofuranyl or morpholinyl.
[0127] In another embodiment, R.sup.2 in formula (Ia) or (Ib)
represents C1 to 6 alkyl substituted by C1 to 6 alkoxy.
[0128] In another embodiment, R.sup.2 in formula (Ia) or (Ib)
represents ethylene or trimethylene substituted by methoxy or
ethoxy.
[0129] When X represents S and Y represents O, a further embodiment
comprises compounds of formula (Ia) or (Ib) wherein R.sup.1
represents hydrogen.
[0130] When X represents S and Y represents O, a yet further
embodiment comprises compounds of formula (Ia) or (Ib) wherein
R.sup.4 represents hydrogen.
[0131] When X represents O and Y represents S, a further embodiment
comprises compounds of formula (Ia) or (Ib) wherein R.sup.1
represents C1 to 6 alkyl.
[0132] When X represents O and Y represents S, a yet further
embodiment comprises compounds of formula (Ia) or (Ib) wherein
R.sup.4 represents C1 to 6 alkyl.
[0133] In one embodiment, the invention relates to the use of
compounds of formula (Ia) or (Ib) wherein X represents S and Y
represents O; R.sup.2 represents optionally substituted C1 to 6
alkyl; and R.sup.1, R.sup.3 and R.sup.4 each represent
hydrogen.
[0134] In one embodiment, the invention relates to the use of
compounds of formula (Ia) or (Ib) herein X represents S and Y
represents O; R.sup.2 represents C1 to 6 alkyl substituted by a
saturated or partially unsaturated 3- to 7-membered ring optionally
incorporating one or two heteroatoms selected independently from O,
N and S, and optionally incorporating a carbonyl group; said ring
being optionally substituted by one or more substituents selected
from halogen, hydroxy, C1 to 6 alkoxy and C1 to 6 alkyl; said alkyl
being optionally further substituted by hydroxy or C1 to 6 alkoxy;
and R.sup.1, R.sup.3 and R.sup.4 each represent hydrogen.
[0135] In one embodiment, the invention relates to the use of
compounds of formula (Ia) or (Ib) wherein X represents S and Y
represents O; R.sup.2 represents C1 to 6 alkyl substituted by Cl to
6 alkoxy; and R.sup.1, R.sup.3 and R.sup.4 each represent
hydrogen.
[0136] In some embodiments, the thioxanthine derivative for use in
the methods of the present embodiments is a compound selected from
the groups consisting of: 1,3-diisobutyl-8-methyl-6-thioxanthine;
1,3-dibutyl-8-methyl-6-thioxanthine;
3-isobutyl-1,8-dimethyl-6-thioxanthine;
3-(2-methylbutyl)-6-thioxanthine;
3-isobutyl-8-methyl-6-thioxanthine; 3-isobutyl-2-thioxanthine;
3-isobutyl-2,6-dithioxanthine; 3-isobutyl-8-methyl-2-thioxanthine;
3-isobutyl-7-methyl-2-thioxanthine;
3-cyclohexylmethyl-2-thioxanthine;
3-(3-methoxypropyl)-2-thioxanthine;
3-cyclopropylmethyl-2-thioxanthine;
3-isobutyl-1-methyl-2-thioxanthine;
3-(2-tetrahydrofuryl-methyl)-2-thioxanthine;
3-(2-methoxy-ethyl)-2-thioxanthine;
3-(3-(1-morpholinyl)-propyl)-2-thioxanthine;
3-(2-furyl-methyl)-2-thioxanthine;
3-(4-methoxybenzyl)-2-thioxanthine;
3-(4-fluorobenzyl)-2-thioxanthine; 3-phenethyl-2-thioxanthine;
(+)-3-(2-tetrahydrofuryl methyl)-2-thioxanthine;
(-)-3-(2-tetrahydrofuryl-methyl)-2-thioxanthine;
3-n-butyl-2-thioxanthine; 3-n-propyl-2-thioxanthine;
3-isobutyl-6-thioxanthine; 2-thioxanthine; and pharmaceutically
acceptable salts thereof.
[0137] With regard to formula (Ia) or (Ib), unless otherwise
indicated, the term "C1 to 6 alkyl" referred to herein denotes a
straight or branched chain alkyl group having from 1 to 6 carbon
atoms. Examples of such groups include methyl, ethyl, 1-propyl,
n-butyl, iso-butyl, tert-butyl, pentyl and hexyl.
[0138] With regard to formula (Ia) or (Ib), term "C1 to 4 alkyl" is
to be interpreted analogously.
[0139] With regard to formula (Ia) or (Ib), the term "C3 to 7
cycloalkyl" referred to herein denotes a cyclic alkyl group having
from 3 to 7 carbon atoms. Examples of such groups include
cyclopropyl, cyclopentyl and cyclohexyl.
[0140] With regard to formula (Ia) or (Ib), unless otherwise
indicated, the term "C1 to 6 alkoxy" referred to herein denotes a
straight or branched chain alkoxy group having from 1 to 6 carbon
atoms. Examples of such groups include methoxy, ethoxy, 1-propoxy,
2-propoxy and tert-butoxy.
[0141] With regard to formula (Ia) or (Ib), term "C1 to 4 alkoxy"
is to be interpreted analogously.
[0142] With regard to formula (Ia) or (Ib), unless otherwise
indicated, the term "halogen" referred to herein denotes fluoro,
chloro, bromo and iodo.
[0143] Examples of a saturated or partially unsaturated 3- to
7-membered ring optionally incorporating one or two heteroatoms
selected independently from O, N and S, and optionally
incorporating a carbonyl group include cyclopropyl, cyclopentyl,
cyclohexyl, cyclopentanone, tetrahydrofuran, pyrrolidine,
piperidine, morpholine, piperazine, pyrrolidinone and piperidinone.
Particular examples include cyclopropyl, cyclohexyl,
tetrahydrofuranyl(tetrahydrofuryl) and morpholinyl.
Derivatives of Pyrrolo[3,2-d]Pyrimidin-4-One Derivatives
[0144] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of
pyrrolo[3,2-d]pyrimidin-4-one.
[0145] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of benzothiophene. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
pyrrolo[3,2-d]pyrimidin-4-one.
[0146] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of benzothiophene. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
pyrrolo[3,2-d]pyrimidin-4-one.
[0147] Derivatives of pyrrolo[3,2-d]pyrimidin-4-one suitable for
use in the methods of the present embodiments are disclosed in U.S.
Pat. No. 7,829,707, incorporated herein by reference in its
entirety.
[0148] In some embodiments, the pyrrolo[3,2-d]pyrimidin-4-one
derivative for use in the methods of the present embodiments is a
compound according to the following Formula II:
##STR00003##
[0149] wherein:
[0150] at least one of X and Y represents S, and the other
represents O or S; L represents a direct bond or C1 to 7 alkylene,
said alkylene optionally incorporating a heteroatom selected from
O, S(O).sub.n and NR.sup.6, said alkylene optionally incorporating
one or two carbon-carbon double bonds, and said alkylene being
optionally substituted by one or more substituents selected
independently from OH, halogen, CN and NR.sup.4R.sup.5, C1 to 6
alkyl and C1 to 6 alkoxy, said alkoxy optionally incorporating a
carbonyl adjacent to the oxygen; n represents an integer 0, 1 or 2;
R.sup.1 represents hydrogen, or
[0151] i) a saturated or partially unsaturated 3 to 7 membered ring
optionally incorporating one or two heteroatoms selected
independently from O, N and S, and optionally incorporating a
carbonyl group, optionally substituted by one or more substituents
independently selected from halogen, SO.sub.2R.sup.9,
SO.sub.2NR.sup.9R.sup.10, OH, C1 to 7 alkyl, C1 to 7 alkoxy, CN,
CONR.sup.2R.sup.3, NR.sup.2COR.sup.3 and COR.sup.3, said alkoxy
being optionally further substituted by C1 to 6 alkoxy and said
alkoxy optionally incorporating a carbonyl adjacent to the oxygen,
and said alkyl being optionally further substituted by hydroxy or
C1 to 6 alkoxy and said alkyl or alkoxy optionally incorporating a
carbonyl adjacent to the oxygen or at any position in the alkyl; or
ii) an aromatic ring system selected from phenyl, biphenyl,
naphthyl or a monocyclic or bicyclic heteroaromatic ring structure
containing 1 to 3 heteroatoms independently selected from O, N and
S, said aromatic ring system being optionally substituted by one or
more substituents independently selected from halogen,
SO.sub.2R.sup.9, SO.sub.2NR.sup.9R.sup.10, OH, C1 to 7 alkyl, C1 to
7 alkoxy, CN, CONR.sup.2R.sup.3, NR.sup.2COR.sup.3 and COW; said
alkoxy being optionally further substituted by C1 to 6 alkoxy and
said alkoxy optionally incorporating a carbonyl adjacent to the
oxygen, and said alkyl being optionally further substituted by
hydroxy or C1 to 6 alkoxy and said alkyl or alkoxy optionally
incorporating a carbonyl adjacent to the oxygen or at any position
in the alkyl; R.sup.12 represents hydrogen or halogen or a carbon
optionally substituted with one to three halogen atoms; at each
occurrence, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.9
and R.sup.10 independently represent hydrogen, C1 to 6 alkyl or C1
to 6 alkoxy said alkoxy optionally incorporating a carbonyl
adjacent to the oxygen, said alkyl being optionally further
substituted by halogen, C1 to 6 alkoxy, CHO, C2 to 6 alkanoyl, OH,
CONR.sup.7R.sup.8 and NR.sup.7COR.sup.8; or the groups
NR.sup.2R.sup.3, NR.sup.4R.sup.5 and NR.sup.9R.sup.10 each
independently represent a 5 to 7 membered saturated azacyclic ring
optionally incorporating one additional heteroatom selected from O,
S and NR.sup.11, said ring being optionally further substituted by
halogen, C1 to 6 alkoxy, CHO, C2 to 6 alkanoyl, OH,
CONR.sup.7R.sup.8 and NR.sup.7COR.sup.8; at each occurrence
R.sup.7, R.sup.8 and R.sup.11 independently represent hydrogen or
C1 to 6 alkyl, or the group NR.sup.7R.sup.8 represents a 5- to
7-membered saturated azacyclic ring optionally incorporating one
additional heteroatom selected from O, S and NR.sup.11; and
pharmaceutically acceptable salts thereof.
[0152] The compounds of formula (II) may exist in enantiomeric
forms. It is to be understood that all enantiomers, diastereomers,
racemates, tautomers and mixtures thereof are included within the
scope of the present embodiments.
[0153] The compounds of formula (II) may exist in tautomeric forms.
All such tautomers and mixtures of tautomers are included within
the scope of the present embodiments.
[0154] With regard to Formula II, unless otherwise indicated, the
term "C1 to 6 alkyl" referred to herein denotes a straight or
branched chain alkyl group having from 1 to 6 carbon atoms.
Examples of such groups include, but are not limited to, methyl,
ethyl, 1-propyl, n-butyl, iso-butyl, tert-butyl, pentyl and
hexyl.
[0155] With regard to Formula II, the term "C1 to 7 alkyl" is to be
interpreted analogously.
[0156] With regard to Formula II, unless otherwise indicated, the
term "C1 to 7 alkylene" referred to herein denotes a straight or
branched chain alkyl group having from 1 to 7 carbon atoms having
two free valencies. Examples of such groups include, but are not
limited to, methylene, ethylene, propylene, hexamethylene and
ethylethylene.
[0157] With regard to Formula II, the term "C1 to 3 alkylene" is to
be interpreted analogously.
[0158] With regard to Formula II, unless otherwise indicated, the
term "C1 to 6 alkoxy" referred to herein denotes a straight or
branched chain alkoxy group having from 1 to 6 carbon atoms.
Examples of such groups include, but are not limited to, methoxy,
ethoxy, 1-propoxy, 2-propoxy(iso-propoxy), tert-butoxy and
pentoxy.
[0159] With regard to Formula II, the term "C1 to 7 alkoxy" is to
be interpreted analogously.
[0160] With regard to Formula II, unless otherwise indicated, the
term "C2 to 6 alkanoyl" referred to herein denotes a straight or
branched chain alkyl group having from 1 to 5 carbon atoms with
optional position on the alkyl group by a carbonyl group. Examples
of such groups include, but are not limited to, acetyl, propionyl
and pivaloyl.
[0161] With regard to Formula II, unless otherwise indicated, the
term "halogen" referred to herein denotes fluoro, chloro, bromo and
iodo.
[0162] Examples of a saturated or partially unsaturated 3- to
7-membered ring optionally incorporating one or two heteroatoms
selected independently from O, N and S, and optionally
incorporating a carbonyl group includes, but is not limited to,
cyclopropane, cyclopentane, cyclohexane, cyclohexene,
cyclopentanone, tetrahydrofuran, pyrrolidine, piperidine,
tetrahydropyridine, morpholine, piperazine, pyrrolidinone and
piperidinone.
[0163] Examples of a monocyclic or bicyclic heteroaromatic ring
structure containing 1 to 3 heteroatoms independently selected from
O, N and S includes, but is not limited to, furan, thiophene,
pyrrole, oxazole, isoxazole, thiazole, imidazole, pyrazole,
triazole, tetrazole, pyridine, pyrazine, pyrimidine, pyridazine,
benzofuran, indole, isoindole and benzimidazole.
[0164] Examples of a 5 to 7 membered saturated azacyclic ring
optionally incorporating one additional heteroatom selected from O,
S and NR.sup.11 includes, but is not limited to, pyrrolidine,
piperidine, piperazine, morpholine and thiomorpholine.
[0165] In the definition of L, "C1 to 7 alkylene; said alkylene
optionally incorporating a heteroatom selected from O, S(O).sub.n
and NR.sup.6; said alkylene optionally incorporating one or two
carbon-carbon double bonds" embraces a saturated or unsaturated
straight or branched chain arrangement of 1 to 7 carbon atoms
having two free valencies and in which any two singly bonded carbon
atoms are optionally separated by O, S or NR.sup.6. The definition
thus includes, for example, methylene, ethylene, propylene,
hexamethylene, ethylethylene, --CH.sub.2.dbd.CH.sub.2--,
CH.sub.2CH.dbd.CH--CH.sub.2--, --CH(CH.sub.3).dbd.CH.sub.2--,
--CH.sub.2.dbd.CH.sub.2--CH.sub.2O--, --CH.sub.2O--,
--CH.sub.2CH.sub.2O--CH.sub.2--,
--CH.sub.2CH.sub.2O--CH.sub.2--CH.sub.2--, --CH.sub.2CH.sub.2S--
and --CH.sub.2CH.sub.2NR.sup.6--.
[0166] In one embodiment, R.sup.1 represents hydrogen.
[0167] In another embodiment, X represents S and Y represents
O.
[0168] In yet another embodiment, Y represents S and X represents
O.
[0169] In yet another embodiment, L is a direct bond or represents
C1 to 7 alkylene, said alkylene optionally incorporating a
heteroatom selected from O, S(O).sub.n and NR.sup.6, said alkylene
optionally incorporating one or two carbon-carbon double bonds, and
said alkylene being optionally substituted by one or more
substituents selected independently from OH, C1 to 6 alkoxy,
halogen, CN and NR.sup.4R.sup.5.
[0170] In yet another embodiment, L is a direct bond or represents
C1 to 7 alkylene; said alkylene being optionally substituted by one
or more substituents selected independently from OH, C1 to 6
alkoxy, halogen, CN and NR.sup.4R.sup.5.
[0171] In yet another embodiment, L is a direct bond or represents
C1 to 7 alkylene; said alkylene being optionally substituted by one
or more C1 to 6 alkoxy.
[0172] In yet another embodiment, L is a direct bond or represents
C1 to 3 alkylene; said alkylene being optionally substituted by one
or more substituents selected independently from OH, C1 to 6
alkoxy, halogen, CN and NR.sup.4R.sup.5.
[0173] In yet another embodiment, L represents C1 to 3 alkylene;
said alkylene being optionally substituted by one or more C1 to 6
alkoxy.
[0174] In yet another embodiment, L is a direct bond or represents
optionally substituted methylene (--CH.sub.2--).
[0175] In yet another embodiment, L is a direct bond or represents
optionally substituted ethylene (--CH.sub.2CH.sub.2--).
[0176] In yet another embodiment, R.sup.1 represents a saturated or
partially unsaturated 3 to 7 membered ring optionally incorporating
one or two heteroatoms selected independently from 0, N and S, and
optionally incorporating a carbonyl group, said ring being
optionally substituted by one or more substituents independently
selected from halogen, SO.sub.2R.sup.9, SO.sub.2NR.sup.9R.sup.10,
OH, C1 to 6 alkyl, C1 to 6 alkoxy, CN, CONR.sup.2R.sup.3,
NR.sup.2COR.sup.3 and COR.sup.3, said alkoxy being optionally
further substituted by C1 to 6 alkoxy; and said alkyl being
optionally further substituted by hydroxy or C1 to 6 alkoxy.
[0177] In yet another embodiment, R.sup.1 represents a saturated or
partially unsaturated 3 to 7 membered ring optionally incorporating
one or two heteroatoms selected independently from 0, N and S, and
optionally incorporating a carbonyl group; said ring being
optionally substituted by one or more substituents independently
selected from halogen, C1 to 6 alkyl and C1 to 6 alkoxy, said
alkoxy being optionally further substituted by C1 to 6 alkoxy.
[0178] In yet another embodiment, R.sup.1 represents an aromatic
ring system selected from phenyl, biphenyl, naphthyl or a
monocyclic or bicyclic heteroaromatic ring structure containing 1
to 3 heteroatoms independently selected from O, N and S, said
aromatic ring being optionally substituted by one or more
substituents independently selected from halogen, SO.sub.2R.sup.9,
SO.sub.2NR.sup.9R.sup.10, OH, C1 to 6 alkyl, C1 to 6 alkoxy, CN,
CONR.sup.2R.sup.3, NR.sup.2COR.sup.3 and COR.sup.3, said alkoxy
being optionally further substituted by C1 to 6 alkoxy, and said
alkyl being optionally further substituted by hydroxy or C1 to 6
alkoxy.
[0179] In yet another embodiment, R.sup.1 represents an aromatic
ring system selected from phenyl, biphenyl, naphthyl or a five- or
six-membered heteroaromatic ring containing 1 to 3 heteroatoms
independently selected from O, N and S, said aromatic ring being
optionally substituted by one or more substituents independently
selected from halogen, C1 to 6 alkyl and C1 to 6 alkoxy, said
alkoxy being optionally further substituted by C1 to 6 alkoxy.
[0180] In yet another embodiment, R.sup.1 represents an optionally
substituted phenyl.
[0181] In yet another embodiment, R.sup.1 represents an optionally
substituted pyridyl.
[0182] In yet another embodiment, L represents C1 to 7 alkylene and
R.sup.1 represents H.
[0183] In yet another embodiment, L represents an optionally
substituted C1 to 3 alkylene and R.sup.1 represents a saturated or
partially unsaturated 3- to 7-membered ring optionally
incorporating one or two heteroatoms selected independently from O,
N and S, and optionally incorporating a carbonyl group, said ring
being optionally substituted by one or more substituents
independently selected from halogen, SO.sub.2R.sup.9,
SO.sub.2NR.sup.9R.sup.10, OH, C1 to 6 alkyl, C1 to 6 alkoxy, CN,
CONR.sup.2R.sup.3, NR.sup.2COR.sup.3 and COR.sup.3, said alkoxy
being optionally further substituted by C1 to 6 alkoxy, and said
alkyl being optionally further substituted by hydroxy or C1 to 6
alkoxy.
[0184] In yet another embodiment, L represents an optionally
substituted C1 to 3 alkylene and R.sup.1 represents a saturated or
partially unsaturated 3- to 7-membered ring optionally
incorporating one or two heteroatoms selected independently from O,
N and S, and optionally incorporating a carbonyl group, said ring
being optionally substituted by one or more substituents
independently selected from halogen, C1 to 6 alkyl and C1 to 6
alkoxy, said alkoxy being optionally further substituted by C1 to 6
alkoxy.
[0185] In yet another embodiment, L represents optionally
substituted C1 to 3 alkylene and R.sup.1 represents an aromatic
ring system selected from phenyl, biphenyl, naphthyl or a five- or
six-membered heteroaromatic ring containing 1 to 3 heteroatoms
independently selected from O, N and S; said aromatic ring being
optionally substituted by one or more substituents independently
selected from halogen, SO.sub.2R.sup.9, SO.sub.2NR.sup.9R.sup.10,
OH, C1 to 6 alkyl, C1 to 6 alkoxy, CN, CONR.sup.2R.sup.3,
NR.sup.2COR.sup.3 and COR.sup.3, said alkoxy being optionally
further substituted by C1 to 6 alkoxy, and said alkyl being
optionally further substituted by hydroxy or C1 to 6 alkoxy.
[0186] In yet another embodiment, L represents optionally
substituted C1 to 3 alkylene and R.sup.1 represents an aromatic
ring system selected from phenyl, biphenyl, naphthyl or a five- or
six-membered heteroaromatic ring containing 1 to 3 heteroatoms
independently selected from O, N and S, said aromatic ring being
optionally substituted by one or more substituents independently
selected from halogen, C1 to 6 alkyl and C1 to 6 alkoxy, said
alkoxy being optionally further substituted by C1 to 6 alkoxy.
[0187] In yet another embodiment, X represents S, Y represents O, L
represents optionally substituted C1 to 3 alkylene and R.sup.1
represents optionally substituted phenyl.
[0188] In yet another embodiment, X represents S, Y represents O, L
represents optionally substituted C1 to 3 alkylene and R.sup.1
represents optionally substituted pyridyl.
[0189] In yet another embodiment, X represents S, Y represents O, L
represents C1 to 3 alkylene, substituted with C1 to 6 alkoxy and
R.sup.1 represents hydrogen.
[0190] Particular compounds of the invention include:
1-butyl-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4-one;
1-isobutyl-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4-one;
1-(pyridin-2-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidi-
n-4-one;
1-(2-fluoro-benzyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyr-
imidin-4-one;
1-[2-(2-methoxyethoxy)-3-propoxybenzyl]-2-thioxo-1,2,3,5-tetrahydro-pyrro-
lo[3,2-d]pyrimidin-4-one;
1-(6-ethoxy-pyridin-2-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d-
-]pyrimidin-4-one;
1-piperidin-3-ylmethyl-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidi-
n-4-one;
1-butyl-4-thioxo-1,3,4,5-tetrahydro-2H-pyrrolo[3,2-d]pyrimidin-2--
one;
1-(2-isopropoxyethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrim-
idin-4-one;
1-(2-methoxy-2-methylpropyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]py-
rimidin-4-one;
1-(2-ethoxy-2-methylpropyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyr-
imidin-4-one;
1-(piperidin-4-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimi-
din-4-one;
1-[(1-methylpiperidin-3-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro--
pyrrolo[3,2-d]pyrimidin-4-one;
1-[2-hydroxy-2-(4-methoxyphenyl)ethyl]-2-thioxo-1,2,3,5-tetrahydro-pyrrol-
o[3,2-d]pyrimidin-4-one;
1-(2-methoxybenzyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4-
-one;
1-(3-methoxybenzyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimi-
din-4-one;
1-(2,4-dimethoxybenzyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-
-d]pyrimidin-4-one;
1-[(3-chloropyridin-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2--
d]pyrimidin-4-one;
1-{[3-(2-ethoxyethoxy)pyridin-2-yl]methyl}-2-thioxo-1,2,3,5-tetrahydro-py-
rrolo[3,2-d]pyrimidin-4-one;
1-[(6-oxo-1,6-dihydropyridin-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyr-
rolo[3,2-d]pyrimidin-4-one;
1-(1H-indol-3-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimid-
in-4-one;
1-(1H-benzimidazol-2-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrro-
lo[3,2-d]pyrimidin-4-one;
1-[(5-chloro-1H-indol-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,-
-2-d]pyrimidin-4-one;
1-[(5-fluoro-1H-indol-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,-
-2-d]pyrimidin-4-one;
1-(1H-indol-6-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimid-
in-4-one;
1-(1H-indol-5-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2--
d]pyrimidin-4-one;
1-[(5-fluoro-1H-indol-3-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,-
-2-d]pyrimidin-4-one;
1-(1H-imidazol-5-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyri-
midin-4-one;
1-(1H-imidazol-2-ylmethyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyri-
midin-4-one;
1-[(5-chloro-1H-benzimidazol-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-pyr-
rolo[3,2-d]pyrimidin-4-one;
1-[(4,5-dimethyl-1H-benzimidazol-2-yl)methyl]-2-thioxo-1,2,3,5-tetrahydro-
-pyrrolo[3,2-d]pyrimidin-4-one;
7-bromo-1-isobutyl-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4--
one; and
1-(3-chlorophenyl)-2-thioxo-1,2,3,5-tetrahydro-pyrrolo[3,2-d]pyri-
midin-4-one; and pharmaceutically acceptable salts thereof.
Derivatives of Benzothiophenes
[0191] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of benzothiophene.
[0192] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of benzothiophene. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
benzothiophene.
[0193] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of benzothiophene. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
benzothiophene.
[0194] Derivatives of benzothiophenes include droloxifene and
raloxifene. Derivatives of benzothiophenes include
2-phenyl-3-aroylbenzothiophene derivatives. Derivatives of
benzothiophene suitable for use in the methods of the present
embodiments are disclosed in U.S. Pat. No. 5,708,009, U.S. Pat. No.
5,719,190, and European Patent No. EP0664125, each of which are
incorporated herein by reference in their entireties.
[0195] In some embodiments, the benzothiophene derivative for use
in the methods of the present embodiments is a compound according
to following Formula (III):
##STR00004##
[0196] wherein R.sup.1 and R.sup.3 are independently hydrogen,
--CH.sub.3,
##STR00005##
[0197] wherein Ar is optionally substituted phenyl; R.sup.2 is
selected from the group consisting of pyrrolidino,
hexamethyleneimino, and piperidino; and pharmaceutically acceptable
salts and solvates thereof.
[0198] Raloxifene is the hydrochloride salt of a compound of
formula III wherein R.sup.1 and R.sup.3 are hydrogen and R.sup.2 is
1-piperidinyl.
[0199] In some embodiments, the benzothiophene derivative for use
in the methods of the present embodiments is a compound according
to following Formula (IIIa):
##STR00006##
[0200] wherein R.sup.1 and R.sup.2 may be the same or different
provided that, when R.sup.1 and R.sup.2 are the same, each is a
methyl or ethyl group, and, when R.sup.1 and R.sup.2 are different,
one of them is a methyl or ethyl group and the other is a benzyl
group or a pharmaceutically acceptable salt thereof. A preferred
salt is the citrate salt.
[0201] A preferred formula I compound is that in which R.sup.1 and
R.sup.2 each are methyl. This preferred compound is known as
droloxifene,
(E)-1-[4'-(2-Dimethylaminoethoxy)phenyl]-1-(3-hydroxyphenyl)-2-phenylbut--
I-ene.
Derivatives of Flavonoids
[0202] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of flavonoid.
[0203] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of flavonoid. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
flavonoid.
[0204] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of flavonoid. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
flavonoid.
[0205] Derivatives of flavonoids include quercetin.
Derivatives of Fluoroindoles
[0206] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of fluoroindole.
[0207] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of fluoroindole. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
fluoroindole.
[0208] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of fluoroindole. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
fluoroindole.
[0209] In some embodiments, the derivative of fluoroindole is
3-(aminoalkyl)-5-fluoroindole 3-(aminoalkyl)-5-fluoroindole or
analogue. See Soubhye et al., J Med Chem. 2010 Dec. 23;
53(24):8747-59, incorporated herein by reference in its
entirety.
Derivatives of Fluorotryptamines
[0210] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a derivative of fluorotryptamine.
[0211] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of fluorotryptamine. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
fluorotryptamine.
[0212] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a derivative of fluorotryptamine. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a derivative of
fluorotryptamine.
[0213] In some embodiments, the derivative of fluorotryptamine is
5-fluorotryptamine.
Sugar Mimetics
[0214] According to some embodiments, the inhibitor of E-selectin
receptor/ligand interactions suitable for use in the methods of the
present embodiments is a Sialyl Lewis X (SLe.sup.x) or
SLe.sup.x-like construct (or SLe.sup.x analog or mimetic), or
Sialyl Lewis A (SLe.sup.a) or SLe.sup.a-like construct. Inhibitors
of E-selectin receptor/ligand interactions are disclosed in U.S.
Pat. No. 5,972,625, incorporated herein by reference in its
entirety.
[0215] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is an SLe.sup.x or SLe.sup.x-like construct
and/or an SLe.sup.a or SLe.sup.a-like construct. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is an SLe.sup.x or
SLe.sup.x-like construct and/or an SLe.sup.a or SLe.sup.a-like
construct.
[0216] In some embodiments, the inhibitor of E-selectin
receptor/MPO-EL interactions suitable for use in the methods of the
present embodiments is a SLe.sup.x or SLe.sup.x-like construct
and/or an SLe.sup.a or SLe.sup.a-like construct. In some
embodiments, the inhibitor of MPO enzymatic activity suitable for
use in the methods of the present embodiments is a SLe.sup.x or
SLe.sup.x-like construct and/or an SLe.sup.a or SLe.sup.a-like
construct.
[0217] The term "selectin" is employed to designate a general class
of receptor which displays a selective adhesive function and which
includes a lectin-like domain responsible for such selective
adhesive function. E-selectin corresponds to glycoprotein ELAM-1
(endothelial leukocyte adhesion molecule-1).
[0218] Sialyl Lewis X (SLe.sup.x) mediates binding of neutrophils
to vascular endothelial cells by binding to E-selectin. (M.
Phillips, et al., Science. 1990, 250, 1130; J. Lowe, et al, Cell.
1990, 63, 475; T. Feizi, Trends. Biochem. Sci. 1991, 16, 84; M.
Tiemeyer., et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1138; L.
Lasky. Science. 1992, 258, 964; and T. Springer, L. A. Lasky,
Nature 1991, 349, 196.) Sialyl Lewis X (SLe.sup.x) is a cell
surface carbohydrate ligand found on neutrophils, anchored onto the
outer membrane thereof by integral membrane glycoproteins and/or
glycolipids. Administration of SLe.sup.x inhibits the
SLe.sup.x/E-selectin interaction and blocks adhesion of neutophils
to endothelial cells. (M. Buerke, et al., J. Clin. Invest., 1994,
1140.). Neutrophil-mediated inflammatory diseases may be treated by
administration of Sialyl Lewis X (SLe.sup.x). SLe.sup.x mimetics
are disclosed in U.S. Pat. No. 5,830,871 and U.S. Pat. No.
5,858,994, incorporated herein by reference in their entireties.
SLe.sup.a is an isomer of SLe.sup.x that also functions as a
prototypical E-selectin binding glycan.
[0219] DeFrees et al., J. Am. Chem. Soc., 117:66-79 (1995) reported
on the in vitro inhibition of binding between E-selectin and
SLe.sup.x-bearing HL-60 cells for a number of SLe.sup.x-related
materials including SLe.sup.x itself, an ethyl glycoside of the
above pentamer and a number of bivalent SLe.sup.x analogs.
[0220] Two SLe.sup.x mimetics synthesized by Uchiyama et al. are of
particular note because they exhibit activities similar to
SLe.sup.x in the E-selectin binding assay. (T. Uchiyama, et al. J.
Am. Chem. Soc. 1995, 117, 5395.) For active natural products
inhibiting E-selectin, see Narasinga Rao, et al., J. Biol. Chem.,
269:19663 (1994).
[0221] The key structural features of SLe.sup.x required for
recognition by E-selectin have been determined by structural and
conformational studies and by comparative studies of the blocking
activity of SLe.sup.x analog families. (B. Brandley, Glycobiology
1993, 3, 633; S. DeFrees, J. Am. Chem. Soc. 1993, 115, 7549; J.
Ramphal, J. Med. Chem. 1994, 37, 3459; D. Tyrrell, Proc. Natl.
Acad. Sci. USA 1991, 88, 10372; R. Nelson. J. Clin. Invest. 1993,
91, 1157; and A. Giannis, Angew. Chem. Int. Ed. Engl. 1994. 33.
178.) The solution conformation of SLe.sup.x has been characterized
using physical methodologies. (Y. C. Lin, et al., J. Am. Chem. Soc.
1992, 114, 5452; Y. Ichikawa, et al. J. Am. Chem. Soc., 1992, 114,
9283; and G. E. Ball et al., J. Am. Chem. Soc., 1992, 114, 5449.)
The three-dimensional structure of the human E-selectin has been
characterized by X-ray diffraction. (B. J. Graves, et al.,. Nature,
1994, 367, 532.) It has been found that the L-fucose, D-galactose
(Gal) and sialic acid moieties of SLe.sup.x are the major
components that interact with E-selectin. N-acetylglucosamine unit
appears to act merely as a linker to connect L-fucose and sialyl
galactose. The six functional groups of SLe.sup.x molecule
including the 2-, 3- and 4-OH groups of L-fucose, the 4- and 6-OH
groups of Gal and the --CO.sub.2.sup.- group of sialic acid are
essential for E-selectin recognition.
Methods of Determining MPO Activity
[0222] MPO activity or inhibition of MPO activity may be determined
by any of a variety of standard methods known in the art. See e.g.,
U.S. Publication No. 20110152224, U.S. Pat. No. 7,108,997,
incorporated herein by reference in their entireties. One such
method is a colorimetric-based assay where a chromophore that
serves as a substrate for the peroxidase generates a product with a
characteristic wavelength which may be followed by any of various
spectroscopic methods including UV-visible or fluorescence
detection. Additional details of colorimetric based assays can be
found in Kettle, A. J. and Winterbourn, C. C. (1994) Methods in
Enzymology. 233: 502-512; and Klebanoff, S. J., Waltersdorph, A. N.
and Rosen, H. (1984) Methods in Enzymology. 105: 399-403, both of
which are incorporated herein by reference. An article by Gerber,
Claudia, E. et al, entitled "Phagocytic Activity and Oxidative
Burst of Granulocytes in Persons with Myeloperoxidase Deficiency"
published in 1996 in Eur. J. Clin. Chem Clin Biochem 34:901-908,
describes a method for isolation for polymorphonuclear leukocytes
(i.e. neutrophils) and measurement of myeloperoxidase activity with
a colorimetric assay, which involves oxidation of the chromgen
4-chloro-1-naphthol.
[0223] Peroxidase activity may be determined by in situ peroxidase
staining in MPO containing cells with flow cytometry-based methods.
Such methods allow for high through-put screening for peroxidase
activity determinations in leukocytes and subpopulations of
leukocytes. An example is the cytochemical peroxidase staining used
for generating white blood cell count and differentials with
hematology analyzers based upon peroxidase staining methods. For
example, the Advia 120 hematology system by Bayer analyzes whole
blood by flow cytometry and performs peroxidase staining of white
blood cells to obtain a total white blood cell count (CBC) and to
differentiate amongst the various white blood cell groups.
[0224] With these methods, whole blood enters the instrument and
red blood cells are lysed in a lysis chamber. The remaining white
blood cells are then fixed and stained in situ for peroxidase
activity. The stained cells are channeled into the flow cytometer
for characterization based upon the intensity of peroxidase
staining and the overall size of the cell, which is reflected in
the amount of light scatter of a given cell. These two parameters
are plotted on the x and y axis, respectively, by conventional flow
cytometry software, and clusters of individual cell populations are
readily discernible. These include, but are not limited, to
neutrophils, monocytes and eosinophils, the three major leukocyte
populations containing visible peroxidase staining.
[0225] During the course of these analyses, leukocytes such as
monocytes, neutrophils, eosinophils and lymphocytes are identified
by the intensity of peroxidase staining and their overall size.
Information about the overall peroxidase activity staining within
specific cell populations is thus inherent in the position of
individual cell clusters (e.g neutrophil, monocyte, eosinophil
clusters) and peroxidase levels within specific cell populations
may be determined. Peroxidase activity/staining in this detection
method is compared to a peroxidase stain reference or calibrant.
Individuals with higher levels of peroxidase activity per leukocyte
are identified by having a cell population whose location on the
cytogram indicates higher levels of peroxidase (i.e., average
peroxidase activity per leukocyte) or by demonstrating a
sub-population of cells within a cell cluster (e.g. neutrophil,
monocyte, eosinophil clusters) which contain higher levels of
peroxidase activity either on average or in a higher subgroup, such
as the higher tertile or quartile.
DEFINITIONS
[0226] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent
from the following detailed description and claims.
[0227] For the purposes of promoting an understanding of the
embodiments described herein, reference will be made to preferred
embodiments and specific language will be used to describe the
same. The terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention. As used throughout this disclosure, the
singular forms "a," "an," and "the" include plural reference unless
the context clearly dictates otherwise. Thus, for example, a
reference to "a composition" includes a plurality of such
compositions, as well as a single composition, and a reference to
"a therapeutic agent" is a reference to one or more therapeutic
and/or pharmaceutical agents and equivalents thereof known to those
skilled in the art, and so forth. Thus, for example, a reference to
"a hostcell" includes a plurality of such host cells, and a
reference to "an antibody" is a reference to one or more antibodies
and equivalents thereof known to those skilled in the art, and so
forth.
[0228] The term "inhibit" includes its generally accepted meaning
which includes prohibiting, preventing, restraining, and slowing,
stopping or reversing progression, severity or a resultant symptom.
As such, the present method includes both medical therapeutic
and/or prophylactic administration, as appropriate.
[0229] Particular embodiments of this invention embrace the use of
inhibitory agents that selectively block the interaction or binding
between an E-selectin receptor and an E-selectin ligand such as MPO
or MPO-EL. As used herein, a "selective inhibitor of the E-selectin
receptor and MPO-EL interaction" or "an agent that selectively
inhibits the interaction between E-selectin receptor and MPO-EL" is
any molecular species that is an inhibitor of the binding between
E-selectin receptor and MPO-EL, but which fails to inhibit, or
inhibits to a substantially lesser degree, the interaction between
E-selectin receptor and other E-selectin ligands.
[0230] As used herein, the term "myeloperoxidase" or "MPO" refers
to a protein that comprises the full-length myeloperoxidase for
given species (e.g. human). The term "myeloperoxidase" or "MPO"
encompasses the novel glycoform disclosed herein, MPO-EL. The
preferred MPO species is human MPO or human MPO-EL.
[0231] As used herein, the term "myeloperoxidase activity" refers
to the turnover or consumption of a substrate based on a
quantifiable amount (e.g., mass) of a MPO. In other words, MPO
activity refers to the amount of MPO needed to convert or change a
substrate into the requisite product in a given time. Methods for
determining or quantifying myeloperoxidase activity are well known
in the art. For example, one method that could be used to determine
myeloperoxidase activity is an immunoassay (such as, for example,
affinity chromatography, immunoelectrophoresis, radioimmunoassay
(RIA), enzyme-linked immunosorbent assays (ELISAs),
immunofluorescent assays, Western blotting, and the like). Such
immunoassays can be homogeneous or heterogeneous immunoassays.
Alternatively, MPO activity can be determined using a
chemiluminescent assay such as that described in U.S. Publication
No. 20090053747, the contents of which are herein incorporated by
reference. Still another method that can be used to determine MPO
activity is a colorimetric-based assay where a chromophore that
serves as a substrate for the peroxidase generates a product with a
characteristic wavelength which may be followed by any of various
spectroscopic methods including UV-visible or fluorescence
detection such as that described in U.S. Pat. No. 7,223,552, the
contents of which are also incorporated by reference in their
entirety.
[0232] Leukocytosis is a raised white blood cell count (the
leukocyte count) above the normal range in the blood. It is
frequently a sign of an inflammatory response. There are five
principle types of leukocytosis: Neutrophilia (the most common
form); Lymphocytosis; Monocytosis; Eosinophilia; and Basophilia. A
leukocyte count above 25 to 30.times.10.sup.9/L is termed a
leukemoid reaction, which is the reaction of a healthy bone marrow
to extreme stress, trauma, or infection. It is different from
leukemia and from leukoerythroblastosis, in which either immature
white blood cells (acute leukemia) or mature, yet non-functional,
white blood cells (chronic leukemia) are present in peripheral
blood.
[0233] Acute myocardial infarction (AMI) refers to a blockage of
one or more of the coronary arteries. Coronary arterial occlusion
due to thrombosis is the cause of most cases of AMI. This blockage
restricts the blood supply to the muscle walls of the heart and is
often accompanied by symptoms such as chest pain, heavy pressure in
the chest, nausea, and shortness of breath, or shooting pain in the
left arm. AMI is accompanied with an inflammatory reaction which
induces cardiac dysfunction and scarring. Rapid restoration of
blood flow to jeopardized myocardium limits necrosis and reduces
mortality.
[0234] "Restenosis" refers to the renewed narrowing of an artery,
e.g. a coronary artery, following a vessel opening or widening
procedure, such as angioplasty or atherectomy. In restenosis, a
vessel that has been treated to at least minimize the volume of a
lesion or blockage and thereby restore blood flow, e.g. by balloon
angioplasty, starts to renarrow, typically within about six months
of the vessel widening procedure. This renarrowing often requires
additional treatment, such as additional angioplasty procedures. It
has been estimated that as much as one third to one half of all
angioplasty procedures are followed by restenosis within the first
six months to one year following the initial vessel widening
procedure.
[0235] In some embodiments, MPO or MPO-EL of the present
embodiments comprises the amino acid sequence of SEQ ID NO: 1, SEQ
ID NO: 2, or SEQ ID NO: 3. In some embodiments, MPO of the present
embodiments comprises an amino acid sequence that is 80% identical
to any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[0236] In some embodiments, MPO or MPO-EL of the present
embodiments comprises an amino acid sequence that is 90% identical
to any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some
embodiments, MPO or MPO-EL of the present embodiments comprises an
amino acid sequence that is 95% identical to any one of SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID NO: 3.
TABLE-US-00001 SEQ ID NO: 1 is as follows:
MGVPFFSSLRCMVDLGPCWAGGLTAEMKLLLALAGLLAILATPQPSEGAAPAVLGEVDTS
LVLSSMEEAKQLVDKAYKERRESIKQRLRSGSASPMELLSYFKQPVAATRTAVRAADYLH
VALDLLERKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTCPEQDKYRTITGMC
NNRRSPTLGASNRAFVRWLPAEYEDGFSLPYGWTPGVKRNGFPVALARAVSNEIVRFPTD
QLTPDQERSLMFMQWGQLLDHDLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKIPPND
PRIKNQADCIPFFRSCPACPGSNITIRNQINALTSFVDASMVYGSEEPLARNLRNMSNQL
GLLAVNQRFQDNGRALLPFDNLHDDPCLLTNRSARIPCFLAGDTRSSEMPELTSMHTLLL
REHNRLATELKSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGPTAMRKYLPTYR
SYNDSVDPRIANVFTNAFRYGHTLIQPFMFRLDNRYQPMEPNPRVPLSRVFFASWRVVLE
GGIDPILRGLMATPAKLNRQNQIAVDEIRERLFEQVMRIGLDLPALNMQRSRDHGLPGYN
AWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNIDIWMGGVSEPLKRKGRVGPL
LACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQISLPRIICDNTGITTVSKNNIFMS
NSYPRDFVNCSTLPALNLASWREAS SEQ ID NO: 2 is as follows:
MGVPFFSSLRCMVDLGPCWAGGLTAEMKLLLALAGLLAILATPQPSEGAAPAVLGEVDTS
LVLSSMEEAKQLVDKAYKERRESIKQRLRSGSASPMELLSYFKQPVAATRTAVRAADYLH
VALDLLERKLRSLWRRPFNVTDVLTPAQLNVLSKSSGCAYQDVGVTCPEQDKYRTITGMC
NNRRSPTLGASNRAFVRWLPAEYEDGFSLPYGWTPGVKRNGFPVALARAVSNEIVRFPTD
QLTPDQERSLMFMQWGQLLDHDLDFTPEPAARASFVTGVNCETSCVQQPPCFPLKIPPND
PRIKNQADCIPFFRSCPACPGSNITIRNQINALTSFVDASMVYGSEEPLARNLRNMSNQL
GLLAVNQRFQDNGRALLPFDNLHDDPCLLTNRSARIPCFLAGDTRSSEMPELTSMHTLLL
REHNRLATELKSLNPRWDGERLYQEARKIVGAMVQIITYRDYLPLVLGPTAMRKYLPTYR
SYNDSVDPRIANVFTNAFRYGHTLIQPFMFRLDNRYQPMEPNPRVPLSRVFFASWRVVLE
GGIDPILRGLMATPAKLNRQNQIAVDEIRERLFEQVMRIGLDLPALNMQRSRDHGLPGYN
AWRRFCGLPQPETVGQLGTVLRNLKLARKLMEQYGTPNNIDIWMGGVSEPLKRKGRVGPL
LACIIGTQFRKLRDGDRFWWENEGVFSMQQRQALAQISLPRIICDNTGITTVSKNNIFMS
NSYPRDFVNCSTLPALNLASWREAS SEQ ID NO: 3 is as follows:
MRLLLGLAGLLAVLIMLQPSEGVPPAVPGEVDTSVVLTCMEEAKRLVDKVYKERRESIKQ
RLHSGLASPMELLSYFKQPVAATRTAVRAADYLHVALSLLERKLRALWPGRFNVTDVLTP
AQLNLLSKTSGCAHQDLGVSCPEKDEYRTITGQCNNRRSPTLGASNRPFVRWLPAEYEDG
FSLPFGWTPRVKRNGFPVPLARAVSNEIVRFPTEKLTPDQQRSLMFMQWGQLLDHDLDFS
PEPAARVSFLTGINCETSCLQQPPCFPLKIPPNDPRIKNQQDCIPFFRSSPACTQSNITI
RNQINALTSFVDASMVYGSEDPLAMRLRNLTNQLGLLAVNTRFQDNGRALLPFDTLRHDP
CRLTNRSANIPCFLAGDSRASEMPELTSMHTLFVREHNRLAKELKRLNAHWNGERLYQEA
RKIVGAMVQIITYRDYLPLVLGREAMRKYLRPYCSYNDSVDPRISNVFTNAFRYGHTLIQ
PFMFRLNSRYQPMQPNPRVPLSRVFFASWRVVLEGGIDPILRGLMATPAKLNRQNQIAVD
EIRERLFEQVMRIGLDLPALNMQRSRDHGLPGYNAWRRFCGLPVPNTVGELGTVLRNLDL
ARRLMKLYQTPNNIDIWIGGVAEPLNKNGRVGPLLACLIGTQFRKLRDGDRFWWQNKGVF
SKKQQQALAKISLPRIICDNTGITFVSKNNIFMSNRFPRDFVRCSRVPALNLAPWRERR
EXAMPLES
[0237] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention. While
the claimed invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one of ordinary skill in the art that various changes and
modifications can be made to the claimed invention without
departing from the spirit and scope thereof. Thus, for example,
those skilled in the art will recognize, or be able to ascertain,
using no more than routine experimentation, numerous equivalents to
the specific substances and procedures described herein. Such
equivalents are considered to be within the scope of this
invention, and are covered by the following claims.
Example 1
G-CSF Induces a Pleiotropic Glycovariant of Myeloperoxidase
[0238] The results summarized in this example provide the first
evidence for functional pleiotropism of myeloperoxidase (MPO), a
well-characterized lysosomal enzyme best known for generation of
cytotoxic oxidants. Specifically, it is shown that G-CSF induces
expression of a cell surface glycoform of MPO that serves as an
E-selectin ligand (MPO-E-selectin Ligand, "MPO-EL"), and that this
molecule is an effector of both leukocyte-endothelial adhesion and
angiotoxicity.
[0239] G-CSF-treated human myeloid cells induce
E-selectin-dependent cytotoxicity in cultured endothelial cells and
depression of cardiac function in a mouse myocardial infarct model.
G-CSF promotes expression of E-selectin ligands on human myeloid
cells, including an uncharacterized .about.65 kDa glycoprotein.
Biochemical studies show that this novel E-selectin ligand is a
catalytically active, sialofucosylated glycoform of myeloperoxidase
(MPO), a well-known lysosomal enzyme. This specialized MPO
glycoform is expressed on circulating G-CSF-mobilized leukocytes,
and is inducible on blood leukocytes and marrow-derived myeloid
cells by G-CSF treatment. Inhibition of MPO activity abrogates
E-selectin-dependent endothelial injury. Disruption of complex
N-linked glycan biosynthesis prevents G-CSF-induced MPO membrane
expression and, concomitantly, blunts angiotoxicity. These findings
define a unique E-selectin ligand and unveil previously unsuspected
MPO functional pleiotropism, placing this lysosomal enzyme at the
nexus of leukocyte migration and vascular pathology.
[0240] Administration of human G-CSF-mobilized leukocytes (ML), but
not human native leukocytes (NL), worsens cardiac function in mice
with surgically induced myocardial infarct. Moreover, co-incubation
of cytokine-stimulated human umbilical vein endothelial cells
(HUVEC) with ML induced robust angiotoxicity that was abrogated by
disruption of E-selectin receptor/ligand interactions. These
findings prompted us to identify the .about.65 kDa E-selectin
ligand specific to G-CSF-mobilized myeloid cells, and to examine
its role(s) in vasculopathy. Biochemical and mass spectrometry
studies reveal that this membrane molecule comprises the heavy
chain of a unique glycovariant of MPO, designated "MPO-EL-selectin
Ligand" (MPO-EL or MPO-EL). We identified that G-CSF treatment of
myeloid cells induces surface MPO expression, concomitant with the
elaboration of specialized N-linked glycan determinants rendering
E-selectin ligand activity. MPO-EL is catalytically active on the
cell membrane and MPO inhibition abrogates myeloid cell
cytotoxicity to vascular endothelium. Heretofore, a leukocyte
surface molecule mediating both leukocyte trafficking and
cytotoxicity has not been identified. Our findings thus reveal
previously unrecognized biologic pleiotropism of both E-selectin
ligands and of MPO, yielding unifying perspectives on the
well-known association between G-CSF administration and vascular
complications and between MPO and vascular inflammatory
conditions.
Results
[0241] Intravascular Administration of Human ML Accentuates Cardiac
Injury in Mice Following Surgically-Induced Myocardial Infarct.
[0242] Administration of G-CSF to patients with acute myocardial
infarct is associated with coronary restenosis and cardiac
depression, and, in patients with coronary artery disease, G-CSF
administration can induce angina pectoris and myocardial infarct.
To directly assess whether human G-CSF-mobilized leukocytes impact
cardiac function following ischemic insult, we administered PBS
(control) and human NL and ML to mice (via jugular vein infusion)
within five hours of surgically-induced myocardial infarct.
Echocardiograms performed three days after infarct showed "stunned"
myocardium, with equivalently dampened cardiac function in all
treatment groups (FIG. 1a) However, compared to day 3 values,
echocardiograms at 7 days post-infarct showed marked improvement in
ejection fraction in mice receiving PBS and NL, whereas mice
injected with ML had sustained profound depression of ejection
fraction (FIG. 1a). These results indicate that administration of
G-CSF-primed leukocytes contributed to significant, irreversible
cardiac injury following myocardial ischemia.
[0243] Assessment of angiotoxicity of NL and ML. Based on the
observed effects of human ML administration in the mouse myocardial
injury model, we sought to determine whether ML alters the
integrity of inflamed endothelium. Accordingly, we incubated
primary human umbilical vein endothelial cells (HUVEC) with
TNF-.alpha. to induce endothelial activation. Stimulated HUVEC were
then incubated in absence of leukocytes, or with addition of NL or
ML. Endothelial cell viability was monitored by trypan blue
exclusion. As shown in FIG. 1b, there was baseline endothelial cell
death after 48 h in TNF-.alpha.-stimulated HUVEC cultures without
leukocyte (No L). Incubation of TNF-.alpha.-stimulated HUVEC with
NL increased endothelial cell death above baseline, and
angiotoxicity was profoundly enhanced by incubation with ML (FIG.
1b).
[0244] Disruption of E-Selectin Receptor/Ligand Interactions Blunts
Angiotoxicity of ML.
[0245] Because we had previously observed that G-CSF induces
E-selectin ligand expression on human myeloid cells, we analyzed
whether angiotoxicity observed in co-culture of stimulated HUVEC
with leukocytes was dependent on E-selectin binding. To this end,
TNF-.alpha.-stimulated HUVEC were incubated with NL, with bone
marrow-derived cells ("BM", comprised predominantly of myeloid
progenitor cells), or with ML in the presence or absence of
function-blocking anti-E-selectin mAb. As shown in FIG. 1c,
incubation of HUVEC with NL and BM cells induced endothelial cell
death, and treatment of HUVEC with function-blocking
anti-E-selectin mAb had no effect on baseline endothelial cell
death (i.e., in absence of leukocytes), nor on angiotoxicity
induced by NL and BM cells. However, the striking increase in
endothelial death observed in co-culture of ML was dramatically
reduced by treatment with anti-E-selectin mAb, to levels observed
in co-culture with NL and BM cells. Altogether, these findings
indicate that in vivo G-CSF treatment heightens myeloid cell
angiotoxicity that is critically dependent on E-selectin
receptor/ligand interactions.
[0246] Identification of the .about.65 kDa E-selectin ligand
induced by G-CSF.
[0247] ML display the E-selectin ligands CLA and HCELL, and an
uncharacterized E-selectin ligand glycoprotein of .about.65 kDa.
Since CLA and HCELL are expressed on immature bone marrow myeloid
cells, and on NL, we reasoned that the enhanced
E-selectin-dependent angiotoxicity observed in ML might be mediated
by the novel .about.65 kDa ligand. To identify this structure, ML
proteins were separated over wheat germ agglutinin (WGA) lectin
columns. The glycoprotein fraction was collected, resolved by
SDS-PAGE, and E-selectin ligands were detected by staining with
E-selectin-Ig chimera (E-Ig). As shown in parallel gel lanes
normalized for input protein content, the WGA chromatography step
preserved and concentrated E-selectin ligands present in ML lysates
(Lanes 1 and 2, FIG. 2a). A series of gel bands were then excised
from the 60-70 kDa region, digested with trypsin and subjected to
MALDI mass spectrometry. In repeated preparations, peptide mass
fingerprinting coupled with bioinformatics analysis identified the
.about.65 kDa protein as the heavy chain of MPO (FIG. 2b). To
confirm the identity of the .about.65 kDa molecule, MPO was
immunoprecipitated (IP) from ML lysates and resolved by SDS-PAGE
under non-reducing or reducing conditions. Western blotting with
anti-MPO mAb revealed only the .about.130-140 kDa homodimer under
non-reducing conditions (FIG. 2c Lane 1). Under reducing
conditions, the precursor at .about.90 kDa and the heavy chain at
.about.65 kDa were stained using anti-MPO mAb 2C7 (FIG. 2c Lane 2),
while the heavy chain was predominantly stained using anti-MPO mAb
3D3 (FIG. 2c Lane 3).
[0248] Analysis of cell surface expression of MPO on ML and NL. MPO
is characteristically stored in native leukocytes within
azurophilic granules. To assess surface expression of MPO on NL and
ML, granulocyte and monocyte fractions from each leukocyte type
were analyzed by flow cytometry. As shown in FIGS. 3a and 3b, NL
granulocytes (NG) and monocytes (NM) express minimal surface MPO,
whereas mobilized granulocytes (MG) and monocytes (MM) display
uniformly high levels of membrane MPO (FIGS. 3a, 3b). To confirm
MPO surface expression, a complementary approach was undertaken
whereby cell membrane proteins from freshly collected NL and ML
were labeled with biotin. MPO immunoprecipitates from lysates of
surface biotinylated cells were resolved by electrophoresis and
blotted with streptavidin-HRP. The western blots revealed
biotinylated MPO prominently in ML, whereas NL showed only trace
amounts on the cell surface (FIG. 3c). Extensive cell washes under
high salt conditions (1.5 M NaCl) did not alter ML surface MPO
levels, nor did digestion of glycosylphosphatidylinisotol (GPI)
anchors with phosphatidylinositol-specific phospholipase C (PI-PLC;
enzyme effectiveness confirmed by loss of CD55, as measured by flow
cytometry), indicating that MPO is an integral membrane component
(data not shown). To determine the activity of surface MPO, ML
membrane proteins labeled with biotin were captured with
streptavidin-conjugated beads and incubated with a peroxidase
substrate. Spectrophotometric analysis revealed a linear
correlation between MPO activity and input cell number (FIG. 3d).
Collectively, these results indicate that in vivo G-CSF treatment
markedly up-regulates expression of catalytically-active MPO on the
surface of mobilized myeloid cells.
[0249] In Vivo G-CSF Administration Induces Expression of MPO-EL,
an E-Selectin Binding Glycoform of MPO.
[0250] E-selectin ligand glycoproteins display sialofucosylated
epitopes reactive with mAb HECA-452. To directly assess whether MPO
serves as an E-selectin ligand, MPO was immunoprecipitated from
lysates of NL and ML, resolved by SDS-PAGE, and blotted with E-Ig.
As shown in FIG. 4a, in immunoprecipitation of lysates normalized
for input cell numbers, MPO from ML showed dramatically more
E-selectin ligand activity than that of NL. Specificity of
E-selectin ligand activity was confirmed by abrogation of E-Ig
staining in the presence of EDTA and by treating immunoprecipitates
with sialidase (data not shown). To determine whether the observed
differences in E-Ig reactivity were resultant from variations in
MPG levels, MPO was immunoprecipitated from equivalent cell numbers
of BM, NL and ML, resolved on SDS-PAGE and blotted with an anti-MPO
Ab which predominantly recognizes the heavy chain (clone 3D3) (FIG.
4b). The blots were then stripped and stained with E-Ig (FIG. 4c).
As shown in FIGS. 4b and 4c, though there was little variation in
MPO staining among the samples, MPO from ML displayed markedly more
E-Ig reactivity than that of BM or NL.
[0251] G-CSF Induces MPO-EL Expression in Myeloid Cells In
Vitro.
[0252] To directly assess whether G-CSF stimulates expression of
MPO-EL, freshly obtained BM, NL and ML were placed in culture and
treated for 48 hours with human G-CSF at 10 ng/ml, a dose
reflecting physiologic concentrations of G-CSF following in vivo
administration.sup.21,37. MPO was immunoprecipitated from
equivalent cell numbers of BM, NL and ML, then resolved on SDS-PAGE
and blotted with an anti-MPO Ab which predominantly recognizes the
heavy chain (clone 3D3) (FIG. 4d); blots were then stripped and
stained with E-Ig (FIG. 4e). As shown in FIG. 4e, G-CSF markedly
induces MPO-EL expression in both BM and NL, without significant
increase in MPO quantity (FIG. 4d). Notably, ex vivo exposure to
G-CSF did not further increase MPO-EL expression in ML (FIG. 4d).
These results provide direct evidence that G-CSF induces MPO-EL
expression on human myeloid cells.
[0253] G-CSF Induces MPO-EL Expression Via N-Glycan-Dependent
Decoration of MPO with HECA-452-Reactive, Sialofucosylated
Determinants.
[0254] Consistent with our prior results.sup.21, flow cytometry
showed that both NL and ML are decorated with HECA-452-reactive
glycans, but ML display markedly increased HECA-452 reactivity
compared to NL (data not shown). To assess HECA-452 expression
relative to surface MPO levels on NL and ML, dual cell surface
staining with HECA-452 and anti-MPO mAb was performed. As shown in
FIG. 5a, there was increased expression, in percent marker-positive
cells and mean channel fluorescence, of both surface MPO and
HECA-452-reactive glycans in ML compared to NL. Cumulative results
(n=15 for NL and ML) for dual marker co-expression are shown in
FIG. 5b.
[0255] To directly examine the effect(s) of G-CSF on cell surface
expression of MPO and HECA-452-reactive sialofucosylations, NL, BM
and ML were treated ex vivo with G-CSF. Changes in membrane
expression of sialofucosylated glycans and MPO were measured by
cell surface staining with HECA-452 and anti-MPO mAb, respectively.
G-CSF treatment of NL and BM cells dramatically increased
expression of surface MPO (FIG. 5c), but, in contrast, ex vivo
G-CSF treatment only modestly increased MPO membrane expression in
ML (FIG. 5c). Flow cytometry analysis showed a significant increase
of HECA452-reactive glycans on NL and BM after G-CSF administration
and only modest increases on ML cells (FIG. 5d).
[0256] Glycoproteins bearing high mannose N-glycans, such as
typically found on lysosomal MPO, require glycosidase-remodeling to
display sialofucosylated determinants on lactosamine scaffolds. To
assess whether such glycan processing is prerequisite for cell
membrane expression of MPO, we employed deoxymannojirimycin (DMJ),
an .alpha.-mannosidase inhibitor that hinders remodeling of high
mannose N-glycans into complex carbohydrates. To this end, BM cells
were treated with neuraminidase (NA) to eliminate existing
E-selectin binding determinants or treated with buffer alone.
Thereafter, cells were cultured with or without G-CSF in the
presence or absence of DMJ to analyze de novo expression of
HECA-452 determinants and of membrane MPO. After 48 h, in the
absence of G-CSF and DMJ, NA-treated IBM cells recovered expression
of HECA-452-reactive glycans to a level approximating that of
buffer treated cells (FIG. 5e, panel 1). Importantly, cells treated
with NA followed by incubation with G-CSF showed marked increased
expression of HECA-452 determinants compared with cells not treated
with G-CSF, whereas cells cultured with G-CSF in the presence of
DMJ markedly diminished the G-CSF-induced expression of
HECA-452-reactive glycans, to levels seen in recovery in the
absence of G-CSF (FIG. 5e, compare panels 2 and 3). These findings
suggest that G-CSF stimulates de novo expression of
HECA-452-reactive moieties predominantly on N-glycans. The residual
HECA-452-determinants displayed in the presence of DMJ likely
reflect the native contribution(s) of sialofucosylated O-linked
glycans found on both glycoproteins and glycolipids. Treatment of
cells with NA did not induce MPO expression (FIG. 5f, panel 1), nor
did pretreatment with NA alter the G-CSF-induced expression of
membrane MPO (FIG. 5f, panel 2). However, G-CSF-induced MPO
expression was significantly attenuated in the presence of DMJ, to
levels equivalent to that of cells not treated with G-CSF (FIG. 5f,
panel 3). G-CSF induces expression of Golgi glycosyltransferases
that synthesize sialofucosylated glycans, showing that surface
expression of MPO-EL is dependent on G-CSF-induced remodeling of
MPO N-glycans into complex sialofucosylated carbohydrates.
[0257] To evaluate the effect of G-CSF on cell surface MPO
enzymatic activity, NL was placed in culture in presence (10 ng/ml)
or absence of G-CSF for 48 hours. Cells were then
surface-biotinylated, lysed, and membrane proteins were
precipitated with streptavidin agarose beads. As shown in FIG. 5h,
ex vivo G-CSF treatment induced surface peroxidase activity.
Addition of DMJ to G-CSF-treated NL prevented induction of surface
enzymatic activity (FIG. 5g). Upon incubation with stimulated
HUVEC, NL treated with G-CSF showed an enhanced cytotoxic effect
compared to that of untreated cells. Importantly, treatment with
G-CSF and DMJ together prevented G-CSF-induced angiotoxicity (FIG.
5h).
[0258] G-CSF-induced myeloid cell angiotoxicity is blunted by
blocking E-selectin receptor/ligand interactions or MPO enzymatic
activity. To assess the effect of MPO-EL expression on endothelial
viability, TNF-.alpha.-stimulated HUVEC were incubated with NL, BM
or ML in the presence or absence of G-CSF. In all HUVEC cultures,
endothelial cell death was markedly increased by co-incubation with
ML or with G-CSF-treated NL or BM, when compared to untreated NL or
BM cells (FIG. 6a). Notably, there was no significant increase in
angiotoxicity with G-CSF-treated ML when compared to untreated ML.
Concomitant incubation with an E-selectin function-blocking
antibody reduced endothelial cell death in ML cultures and in NL or
BM cultures treated with G-CSF. Importantly, incubation with
4-aminobenzoic hydrazide (4-ABAH), a specific MPO inhibitor, most
effectively decreased endothelial cell death in all cultures (FIG.
6a).
[0259] In an alternative approach, we abrogated E-selectin ligand
activity by use of sialidase to cleave terminal sialic acid from
sLex, the canonical E-selectin binding determinant. Incubation of
endothelial cells with sialidase had no effect endothelial
viability; however, co-culture of endothelial cells with ML treated
with sialidase significantly decreased endothelial cell death
compared to that in co-cultures of untreated ML (FIG. 6b). Since ML
treated with neuraminidase exhibit decreased angiotoxicity, we
assessed whether sialidase treatment of ML to disrupt E-selectin
ligand activity would have a beneficial effect on recovery of heart
function after myocardial infarct. To this end, ML or
sialidase-treated ML were injected in mice within five hours after
surgically-induced infarct. Notably, sialidase treatment did not
affect the clearance of infused ML, as no differences in the levels
of circulating cells were noted among treated and untreated ML
(data not shown) for the first 24 hours after administration.
Echocardiogram obtained 7 days post-MI showed improved ejection
fraction in mice injected with neuraminidase-treated ML compared to
that of mice injected with untreated ML (FIG. 6c).
DISCUSSION
[0260] The known association of G-CSF administration and vascular
complications prompted us to investigate whether G-CSF-stimulated
myeloid cells alter vascular integrity. We observed that injection
of G-CSF-mobilized human leukocytes (ML) accentuates cardiac injury
in mice following myocardial ischemia. Also, ML displays heightened
cytotoxicity to vascular endothelium which is blunted by
interruption of E-selectin receptor/ligand interactions.
[0261] Our data indicate that the .about.65 kDa E-selectin ligand
comprises the heavy chain of a specialized, membrane-expressed
glycoform of MPO, a well-characterized lysosomal enzyme whose
synthesis is induced by G-CSF. MPO is initially expressed during
promyelocyte development, and is characteristically stored in
azurophilic granules of neutrophils, monocytes and macrophages.
Mature MPO is a heme-containing glycoprotein of .about.140 kDa
which consists of two catalytically active monomers of .about.75
kDa, each comprised of a 55-65 kDa heavy chain and a 10-15 kDa
light chain, generated from a .about.90 kDa precursor.
[0262] Cell surface presentation is essential for MPO-EL to
function as an E-selectin ligand. Inhibition of N-glycan
Golgi-processing with DMJ abrogated G-CSF-induced decoration of MPO
with HECA452-reactive glycans, resulting in markedly reduced MPO
membrane expression. DMJ did not eliminate the re-expression of
E-selectin ligands following sialidase treatment; it only inhibited
the boost in E-selectin ligand expression induced by G-CSF. DMJ
does not affect processing of O-linked glycans, therefore any
residual HECA-452 reactivity in the presence of DMJ reflects
contribution(s) of O-sialofucosylated glycoproteins and
glycolipids. Thus, through induction of relevant
glycosyltransferases, G-CSF licenses sialofucosylations on N-linked
carbohydrates and creates E-selectin ligands, including MPO-EL.
[0263] The precise mechanism(s) by which carbohydrate modifications
target MPO expression on the cell membrane is uncharacterized at
present. However, there is a well-known correlation between cell
surface expression of glycoproteins and the N-linked glycosylation
pathway which assures insertion and remodeling of correct
carbohydrates on de novo synthesized glycoprotein during their
transit through reticulum endoplastic (RE) and the Golgi. MPO
biosynthesis follows a complex succession of proteolytic cleavages
and glycan remodeling which directs this glycoprotein either to the
azurophilic granules or to the extracellular space. It is believed
that MPO can reach the azurophilic granules either by passing
through the trans-Golgi into the late endosomes or by first being
displayed on the plasma membrane followed by internalization into
endosomes. Under physiological conditions, G-CSF induces MPO
synthesis during development of myelocyte and monocyte precursors
and stops as cells reach maturity. MPO synthesis can be reactivated
in mature leukocytes. Hematopoietic stem/progenitor cell
mobilization with clinical doses of G-CSF induces myeloid cell
MPO-EL synthesis and its expression on plasma membrane positions
this catalytically active enzyme in direct proximity to endothelial
cells. Washing cells with high salt solutions (e.g., 1.5 M NaCl) or
treatment with PI-PLC (to cleave GPI-anchors) did not release MPO
from the cell surface, suggesting that this glycoprotein is
membrane integrated.
[0264] The data presented here show for the first time that G-CSF
promotes a dual function for MPO--as a cytotoxic effector and an
E-selectin ligand--providing a unifying perspective on the
pathobiology of G-CSF-induced vascular complications. Engagement of
vascular E-selectin with corresponding ligands expressed on the
surface of circulating cells initiates decelerative contacts of
circulating cells onto the target endothelium under hemodynamic
flow conditions, thereby allowing for subsequent integrin-mediated
firm adherence and extravasation. The ability of E-selectin to
recruit leukocytes to sites of inflammation guarantees that the
host defense arsenal is delivered to the correct "address".
However, at the outset of recruitment, E-selectin-mediated MPO-EL
binding to endothelium and consequent production of oxidizing
agents would serve to affix toxic metabolites to the endothelium,
thereby heightening vascular injury within inflamed tissue(s).
Notably, sickle cell crises, coronary artery disease,
atherosclerosis, and stroke, have all been linked to MPO. Moreover,
MPO catalyzes oxidation of vascular nitric oxide (NO), resulting in
consumption of NO and formation of highly reactive nitrite species
which participate in deleterious protein tyrosine nitration.
Endogenous NO is a critical anti-inflammatory and anti-atherogenic
factor that inhibits endothelial activation and prevents leukocyte
adhesion by suppressing expression of E-selectin induced by
cytokines such as TNF-.alpha.. Thus, independent of direct
cytotoxicity, through its enzymatic role in reducing NO
bioavailability, MPO sustains expression of E-selectin on
endothelial cells that further supports binding of leukocytes
bearing E-selectin ligands such as MPO-EL. This loop would serve to
compound vascular injury. Importantly, the fact that
life-threatening sickle cell crisis is induced by G-CSF
administration and, specifically, that anti-E-selectin agents can
ameliorate this process, suggests a role for MPO-EL in this
vasculopathy.
[0265] Within the past decade, several clinical trials using G-CSF
to mobilize hematopoietic stem cells in patients with ischemic
heart disease have been performed with the intent to improve
myocardial function. Though generally safe, significant adverse
vascular effects have been observed when G-CSF is administered to
patients with coronary artery disease, especially in patients
receiving G-CSF in the immediate peri-infarct period or in those
with significant ischemic symptoms. Indeed, in post-MI patients
receiving coronary stenting, an alarming rate of restenosis was
observed in those patients that received G-CSF immediately prior to
stenting. Our results showing that administration of human ML to
mice following surgically-induced myocardial infarct augments
myocardial injury, supporting these clinical observations. Notably,
mice with infarcts that received ML treated with sialidase had
relative preservation of ejection fraction, implicating a role for
E-selectin ligand activity in the observed ML-associated myocardial
compromise. These findings, coupled with data from in vitro studies
showing that treatment of myeloid cells with G-CSF increases
angiotoxicity which is reversed by blocking MPO activity and by
disruption of E-selectin binding, directly link MPO-EL expression
with endothelial injury, offering a new operational paradigm for
MPO-associated pathobiology. Leukocyte surface MPO expression is
already known to be associated with development of vasculitides
such as Wegener's granulomatosis and Churg-Strauss syndrome. These
conditions are characterized by expression of MPO on activated
neutrophils which serves as an antigenic target for antineutrophil
cytoplasm autoantibodies (ANCA). Notably, ANCA binding activates
neutrophils, causing increased endothelial adhesion and
angiotoxicity, and, specifically, G-CSF induces flares of
ANCA-associated vasculitis. It is also well-known that MPC) and
MPO-generated hypohalide products are concentrated in
atherosclerotic lesions. These important clinical observations
underscore the novel finding here that MPO, a major effector of
cytotoxicity, is presented on the leukocyte surface, in a form that
localizes this cytocidal agent in direct contact with endothelial
cells. Thus, the results here focus new attention on the
multipurpose role(s) of MPO in inflammatory conditions, and provide
novel mechanistic insights on how interruption of E-selectin
receptor/ligand interactions may serve to prevent G-CSF-induced
vascular complications and, potentially, sickle cell crises,
atherosclerosis, and ANCA-related vasculitic syndromes.
[0266] Materials and Methods
[0267] Cells.
[0268] Human cells were obtained and used in accordance with the
procedures approved by the Human Experimentation and Ethics
Committees of Partners Cancer Care Institutions (Massachusetts
General Hospital, Brigham and Women's Hospital, and Dana Farber
Cancer Institute). ML were collected by blood apheresis from
healthy donors receiving G-CSF to mobilize hematopoietic
progenitors for hematopoietic stem cell transplantation (samples
were provided by the Cell Manipulation Core Facility from Brigham
and Women's Hospital/Dana Farber Cancer Institute). NL were
isolated from blood obtained from healthy volunteers. Human BM
cells were separated from filter sets used during bone marrow
harvests performed at Massachusetts General Hospital. To ensure
consistency in sample preparation and obtain data most relevant to
native human biology, cells from blood and marrow samples were
uniformly processed for relevant analyses within 2 hours of
collection. The leukocytes from different sources were purified by
direct centrifugal sedimentation or by Ficoll Histopaque.RTM.-1077
(Sigma-Aldrich, St. Louis, Mo.) separation and the residual red
cells were eliminated by hypotonic lysis. Isolated cells were
washed with phosphate buffered saline (PBS) and used in downstream
procedures.
[0269] Antibodies.
[0270] Western blot analysis was performed with the following
antibodies: recombinant mouse E-selectin-human Fc chimera, (R&D
Systems, Minneapolis, Minn.), horseradish peroxidase
(HRP)-conjugated goat anti-human Ig, (Southern Biotech, Birmingham,
Ala.), mouse monoclonal anti-MPO, clone 2C7 (Abcam, Cambridge,
Mass.), goat anti-mouse IgG HRP-conjugated and streptavidin
HRP-conjugated (BD Bioscience, San Jose, Calif.). Mouse anti-MPO
mAb, clone 1A1 from Abcam (Cambridge, Mass.), was used for
immunoprecipitations. Flow cytometry analysis was performed with
the following antibodies: HECA-452 mAb (BD Bioscience, San Jose,
Calif.); goat anti-rat IgM fluorescein isothiocyanate
(FITC)-conjugated, rat IgM isotype, and mouse IgG isotype (Southern
Biotech, Birmingham, Ala.); mouse anti-MPO mAb, clone 2C7 (Abcam,
Cambridge, Mass.) and goat anti-mouse IgG phycoeiythrin
(PE)-conjugated (Santa Cruz Biotechnology, Santa Cruz, Calif.).
[0271] Immunoprecipitations, SDS-PAGE and Western Blotting.
[0272] Leukocytes were lysed in Buffer A (0.5 mM Tris, pH=8, 150 mM
NaCl, 20 .mu.g/ml PMSF, 0.02% sodium azide) supplemented with 1%
Triton (Sigma-Aldrich, St. Louis, Mo.) and protease inhibitor
cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Cell lysates
were precleared with rProtein G Agarose beads (Invitrogen,
Carlsbad, Calif.). Immunoprecipitations were performed at 4.degree.
C. for 16 h, by incubating the precleared lysates with 2 .mu.g
antibody and a fresh batch of agarose beads blocked in advance with
1 mg/ml bovine serum albumin (Sigma-Aldrich, St. Louis, Mo.). After
extensive washes, the beads were boiled with sample buffer and the
released antigens or cell lysates were resolved on 7.5% Tris-HCl
SDS-PAGE (Criterion.TM. Precast Gel, Bio-Rad Laboratories,
Hercules, Calif.). In western blot experiments separated proteins
were transferred to Sequi-BlotTMPVDF Membrane (Bio-Rad
Laboratories, Hercules, Calif.) which was blocked for 2 h with a
solution of 5% Non-Fat Milk (LabScientific, Livingston, N.J.).
Proteins were detected with E-selectin-Ig chimera or with anti-MPO
mAb: clone 2C7 (Abcam) and clone 3D3 (a generous gift from Carrie
Rice at Maine Biotechnology Services, ME). Secondary antibodies
goat anti-human IgG and goat anti-mouse IgG, HRP-conjugated were
detected with Lumi-Light Western Blotting Substrate, (Roche
Diagnostics GmbH, Mannheim, Germany).
[0273] Lectin Chromatography and Mass Spectrometry Analysis.
[0274] Wheat germ agglutinin (WGA) lectin chromatography was
employed to purify the glycoprotein pool of selectin ligands from
ML. Cell lysates were incubated with WGA immobilized to agarose
beads (Pierce, Rockford, Ill.) and, after extensive washes, the
glycoproteins were released and dialyzed. Two 7.5% SDS-PAGE were
run in parallel to resolve the selectin ligands. The proteins from
one gel were transferred to a PVDF membrane which was stained in
western blot with E-selectin Ig. The migration pattern of selectin
ligands, revealed by western blot was used to locate the relevant
proteins in the second gel. Thin slices were excised from the gel
area where the selectin ligands migrated. Protein in-gel digestion
was performed with proteomics grade trypsin (Sigma-Aldrich, St.
Louis, Mo.) by covering the gel slices with trypsin solution (40 mM
ammonium bicarbonate in 9% acetonitrile) and incubating at
37.degree. C. for 16 h. Gel pieces were vortexed with 0.1%
trifluoroacetic acid (Fluka) in 50% acetonitrile (Fisher
Scientific, Pittsburgh, Pa.) to extract the peptides, which were
further concentrated with a C18 Zip Tip (Millipore Corporation,
Billerica, Mass.). The peptide mixture was analyzed with a
MALDI-TOF mass spectrometer (Axima-CFR, Kratos-Shimadzu Biotech,
Manchester, UK) using dihydroxybenzoic acid (DHB, 10 mg/mL) as
matrix. The instrument was calibrated with a set of peptide
standards (Proteomass MALDI-MS calibration kit, Sigma).
[0275] Cell Surface Biotinylation.
[0276] Cells were washed with PBS and incubated with
NHS-PEO.sub.4-biotin (Pierce, Rockford, Ill.) or DMSO for negative
control, as recommended in the manufacturer specifications. After
15 min at room temperature, cells were washed with PBS supplemented
with non-essential amino acids followed by extensive wash with PBS.
Biotinylation efficiency was monitored by cell surface staining
with PE-conjugated streptavidin followed by flow cytometry
analysis.
[0277] Flow Cytometry.
[0278] Cell surface expression of E-selectin ligands and MPO was
determined by indirect single-color immunostaining with HECA-452
and anti-MPO (2C7) mAbs, respectively. Cells were incubated with
primary antibodies and their matched isotype controls in PBS with
2% FBS for 20 min, on ice. After successive washes with PBS and 2%
FBS cells were stained with FITC-conjugated secondary antibody for
HECA-452 and PE-conjugated secondary antibody for MPO. Stained
cells were then washed, resuspended in PBS, and analyzed using the
Cytomics FC 500 MPL flow cytometer (Beckman Coulter, Miami,
Fla.).
[0279] In Vitro G-CSF Treatment.
[0280] Myeloid cells (10.sup.6 cells/ml), isolated from different
sources were cultured with RPMI 1640 medium (Mediatech, Inc,
Manassas, Va.) with 10% FBS, 1% pen/strep and 10 ng/ml recombinant
human G-CSF (Neupogen) from Amgen Mfg. Ltd., CA. Cells were
maintained in culture for 72 h, at 37.degree. C. and G-CSF aliquots
were added after each 24 h period.
[0281] Neuraminidase and DMJ Treatment.
[0282] Cells were isolated from the buffy coat of bone marrow
aspirates after centrifugal sedimentation and red blood cell lysis.
Purified nucleated cells (10.sup.7/m1) were incubated with Vibrio
Cholerae neuraminidase (Roche Diagnostics GmbH, Mannheim, Germany),
for 1 h at 37.degree. C. Efficiency of sialic acid removal was
confirmed by cell surface staining with HECA-452 mAb followed by
flow cytometry analysis. After extensive washes, cells were divided
in equal numbers and cultured in RPMI 1640 medium, for 48 h, with
or without G-CSF treatment. In parallel, a subset of cells cultured
with G-CSF was treated with 1 mM deoxymannojirimycin (DMJ). The
effect(s) of G-CSF and DMJ were assessed by flow cytometry analysis
of surface expression of HECA-452 determinants and of MPO.
[0283] Detection of Membrane MPO Activity.
[0284] Aliquots of NL and ML were surface biotinylated, lyesed and
membrane proteins were precipitated with streptavidin-conjugated
agarose beads. The beads were incubated with a chromogenic
peroxidase substrate, o-Phenylenediamine dihydrochioride (OPD)
(Sigma) and the activity of surface MPO was monitored
spectrophotometrically.
[0285] Endothelial Cell Death Evaluation and Inhibition of MPO
Activity Assay.
[0286] HUVECs were obtained from the Vascular Biology Core Facility
of the Department of Pathology of Brigham and Women's Hospital and
were grown on fibronectin (BD Bioscience) coated plates (20
.mu.g/ml) with Medium 199 (Cambrex, East Rutherford, N.J.),
supplemented with 20% FBS, 2 mM L-glutamine (Invitrogen), 1%
pen/strep, 100 .mu.g/ml heparin (Sigma) and 50 .mu.g/m1 endothelial
cell growth supplement (Biomedical Technologies, Stoughton, Mass.).
Endothelial cells were activated for 6 h in culture with 40 ng/ml
TNF.alpha. to express E-selectin and incubation with 10 .mu.g/ml
mouse anti-human CD62E antibody (BD Pharmingen) was used to block
E-selectin function. Leukocytes of different origins were treated
with G-CSF or G-CSF and DMJ and co-cultured with activated HUVECs
for 48 h. To inhibit MPO activity leukocytes were incubated with
100 .mu.M 4-Aminobenzoic hydrazide (4-ABAH) (Sigma-Aldrich) in
culture media for 48 h at 37.degree. C. Endothelial cell death was
quantified by trypan blue exclusion assay and the number of dead
cells was reported as percentage from the number of total cells
counted per squared unit of HUVEC layer.
[0287] Surgically Induced Myocardial Infarct (MI) and
Echocardiographic Measurements.
[0288] Adult RAG-2/JAK-3 SCID mice were maintained on a standard
mouse chow diet and water ad libitum, and housed in a
temperature-controlled environment under an alternating 12-hour
light/dark cycle. All animal handling procedures adhered strictly
to the approved guidelines of the Institutional Animal Care and Use
Committee. Mice paired by weight and sex were distributed in two
groups: in one group experimental MI was induced by permanent left
anterior descending artery (LAD) ligation, as previously
described.sup.67 and another group that underwent open thoracotomy
without coronary ligation (sham operated). Within five hours
following the surgery, mice from each group were injected via
jugular vein either with PBS or different cell types: NL, ML or
sialidase-treated ML (4.times.10.sup.6 cells/mouse). The surgeon
who performed the MI procedure and the jugular vein perfusions was
blinded to the type of cells/PBS injected in each animal.
Echocardiography was performed on all mice before surgery
(baseline), at 3 days and 7 days following surgery using an 18 to
38 MHz linear-array transducer with a digital ultrasound system
(Vevo 2100 Imaging System, VisualSonics, Toronto, Canada). Standard
parasternal long- and short-axis views were obtained during each
echocardiographic examination and image measurements were performed
offline by an investigator blinded to animal groups and the cell
types/PBS injections.
[0289] PI-PLC Cell Treatment.
[0290] ML (1.times.10.sup.6) were incubated in PBS with 1 unit of
PI-PLC from Bacillus cereus (Molecular Probes, OR) for 30 min. at
4.degree. C. To verify the enzyme efficiency cells were stained
with anti-CD55 mAb, clone JS11 (BioLegend, CA) since CD55 is known
to be attached to the hematopoietic cell membrane by a GPI-anchor.
Cells were stained with anti-MPO mAB, clone 2C7 to verify whether
MPO is GPI-anchored or with anti-CD44 mAb, clone 515 as negative
control. Anti-mouse IgG-FITC was used as secondary antibody
staining and cells were monitored by flow cytometry.
[0291] Washes with High Salt Solutions.
[0292] Cells were washed with 0.5, 1 or 1.5 M NaCl by agitation at
room temperature and quickly returned to PBS. Cell surface
expression of MPO was monitored by staining with anti-MPO mAB,
clone 2C7, anti-mouse IgG-FITC secondary antibody and flow
cytometry analysis.
[0293] Statistical Analysis.
[0294] The compared values represent means of cell subsets isolated
from random human clinical samples of multiple donors. The error
bars represent standard deviation (SD). Statistical analysis was
performed using a two-tailed, unpaired Student's t-test of the
means. P values<0.05 were considered statistically
significant.
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Sequence CWU 1
1
31747PRTHomo sapiens 1Met Gly Val Pro Phe Phe Ser Ser Leu Arg Cys
Met Val Asp Leu Gly 1 5 10 15 Pro Cys Trp Ala Gly Gly Leu Thr Ala
Glu Met Lys Leu Leu Leu Ala 20 25 30 Leu Ala Gly Leu Leu Ala Ile
Leu Ala Thr Pro Gln Pro Ser Glu Gly 35 40 45 Ala Ala Pro Ala Val
Leu Gly Glu Val Asp Thr Ser Leu Val Leu Ser 50 55 60 Ser Met Glu
Glu Ala Lys Gln Leu Val Asp Lys Ala Tyr Lys Glu Arg 65 70 75 80 Arg
Glu Ser Ile Lys Gln Arg Leu Arg Ser Gly Ser Ala Ser Pro Met 85 90
95 Glu Leu Leu Ser Tyr Phe Lys Gln Pro Val Ala Ala Thr Arg Thr Ala
100 105 110 Val Arg Ala Ala Asp Tyr Leu His Val Ala Leu Asp Leu Leu
Glu Arg 115 120 125 Lys Leu Arg Ser Leu Trp Arg Arg Pro Phe Asn Val
Thr Asp Val Leu 130 135 140 Thr Pro Ala Gln Leu Asn Val Leu Ser Lys
Ser Ser Gly Cys Ala Tyr 145 150 155 160 Gln Asp Val Gly Val Thr Cys
Pro Glu Gln Asp Lys Tyr Arg Thr Ile 165 170 175 Thr Gly Met Cys Asn
Asn Arg Arg Ser Pro Thr Leu Gly Ala Ser Asn 180 185 190 Arg Ala Phe
Val Arg Trp Leu Pro Ala Glu Tyr Glu Asp Gly Phe Ser 195 200 205 Leu
Pro Tyr Gly Trp Thr Pro Gly Val Lys Arg Asn Gly Phe Pro Val 210 215
220 Ala Leu Ala Arg Ala Val Ser Asn Glu Ile Val Arg Phe Pro Thr Asp
225 230 235 240 Gln Leu Thr Pro Asp Gln Glu Arg Ser Leu Met Phe Met
Gln Trp Gly 245 250 255 Gln Leu Leu Asp His Asp Leu Asp Phe Thr Pro
Glu Pro Ala Ala Arg 260 265 270 Ala Ser Phe Val Thr Gly Val Asn Cys
Glu Thr Ser Cys Val Gln Gln 275 280 285 Pro Pro Cys Phe Pro Leu Lys
Ile Pro Pro Asn Asp Pro Arg Ile Lys 290 295 300 Asn Gln Ala Asp Cys
Ile Pro Phe Phe Arg Ser Cys Pro Ala Cys Pro 305 310 315 320 Gly Ser
Asn Ile Thr Ile Arg Asn Gln Ile Asn Ala Leu Thr Ser Phe 325 330 335
Val Asp Ala Ser Met Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn 340
345 350 Leu Arg Asn Met Ser Asn Gln Leu Gly Leu Leu Ala Val Asn Gln
Arg 355 360 365 Phe Gln Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn
Leu His Asp 370 375 380 Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg
Ile Pro Cys Phe Leu 385 390 395 400 Ala Gly Asp Thr Arg Ser Ser Glu
Met Pro Ile Glu Leu Thr Ser Met 405 410 415 His Thr Leu Leu Leu Arg
Glu His Asn Arg Leu Ala Thr Glu Leu Lys 420 425 430 Ser Leu Asn Pro
Arg Trp Asp Gly Glu Arg Leu Tyr Gln Glu Ala Arg 435 440 445 Lys Ile
Val Gly Ala Met Val Gln Ile Ile Thr Tyr Arg Asp Tyr Leu 450 455 460
Pro Leu Val Leu Gly Pro Thr Ala Met Arg Lys Tyr Leu Pro Thr Tyr 465
470 475 480 Arg Ser Tyr Asn Asp Ser Val Asp Pro Arg Ile Ala Asn Val
Phe Thr 485 490 495 Asn Ala Phe Arg Tyr Gly His Thr Leu Ile Gln Pro
Phe Met Phe Arg 500 505 510 Leu Asp Asn Arg Tyr Gln Pro Met Glu Pro
Asn Pro Arg Val Pro Leu 515 520 525 Ser Arg Val Phe Phe Ala Ser Trp
Arg Val Val Leu Glu Gly Gly Ile 530 535 540 Asp Pro Ile Leu Arg Gly
Leu Met Ala Thr Pro Ala Lys Leu Asn Arg 545 550 555 560 Gln Asn Gln
Ile Ala Val Asp Glu Ile Arg Glu Arg Leu Phe Glu Gln 565 570 575 Val
Met Arg Ile Gly Leu Asp Leu Pro Ala Leu Asn Met Gln Arg Ser 580 585
590 Arg Asp His Gly Leu Pro Gly Tyr Asn Ala Trp Arg Arg Phe Cys Gly
595 600 605 Leu Pro Gln Pro Glu Thr Val Gly Gln Leu Gly Thr Val Leu
Arg Asn 610 615 620 Leu Lys Leu Ala Arg Lys Leu Met Glu Gln Tyr Gly
Thr Pro Asn Asn 625 630 635 640 Ile Asp Ile Trp Met Gly Gly Val Ser
Glu Pro Leu Lys Arg Lys Gly 645 650 655 Arg Val Gly Pro Leu Leu Ala
Cys Ile Ile Gly Thr Gln Phe Arg Lys 660 665 670 Leu Arg Asp Gly Asp
Arg Phe Val Val Trp Glu Asn Glu Gly Val Phe 675 680 685 Ser Met Gln
Gln Arg Gln Ala Leu Ala Gln Ile Ser Leu Pro Arg Ile 690 695 700 Ile
Cys Asp Asn Thr Gly Ile Thr Thr Val Ser Lys Asn Asn Ile Phe 705 710
715 720 Met Ser Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu
Pro 725 730 735 Ala Leu Asn Leu Ala Ser Trp Arg Glu Ala Ser 740 745
2745PRTHomo sapiens 2Met Gly Val Pro Phe Phe Ser Ser Leu Arg Cys
Met Val Asp Leu Gly 1 5 10 15 Pro Cys Trp Ala Gly Gly Leu Thr Ala
Glu Met Lys Leu Leu Leu Ala 20 25 30 Leu Ala Gly Leu Leu Ala Ile
Leu Ala Thr Pro Gln Pro Ser Glu Gly 35 40 45 Ala Ala Pro Ala Val
Leu Gly Glu Val Asp Thr Ser Leu Val Leu Ser 50 55 60 Ser Met Glu
Glu Ala Lys Gln Leu Val Asp Lys Ala Tyr Lys Glu Arg 65 70 75 80 Arg
Glu Ser Ile Lys Gln Arg Leu Arg Ser Gly Ser Ala Ser Pro Met 85 90
95 Glu Leu Leu Ser Tyr Phe Lys Gln Pro Val Ala Ala Thr Arg Thr Ala
100 105 110 Val Arg Ala Ala Asp Tyr Leu His Val Ala Leu Asp Leu Leu
Glu Arg 115 120 125 Lys Leu Arg Ser Leu Trp Arg Arg Pro Phe Asn Val
Thr Asp Val Leu 130 135 140 Thr Pro Ala Gln Leu Asn Val Leu Ser Lys
Ser Ser Gly Cys Ala Tyr 145 150 155 160 Gln Asp Val Gly Val Thr Cys
Pro Glu Gln Asp Lys Tyr Arg Thr Ile 165 170 175 Thr Gly Met Cys Asn
Asn Arg Arg Ser Pro Thr Leu Gly Ala Ser Asn 180 185 190 Arg Ala Phe
Val Arg Trp Leu Pro Ala Glu Tyr Glu Asp Gly Phe Ser 195 200 205 Leu
Pro Tyr Gly Trp Thr Pro Gly Val Lys Arg Asn Gly Phe Pro Val 210 215
220 Ala Leu Ala Arg Ala Val Ser Asn Glu Ile Val Arg Phe Pro Thr Asp
225 230 235 240 Gln Leu Thr Pro Asp Gln Glu Arg Ser Leu Met Phe Met
Gln Trp Gly 245 250 255 Gln Leu Leu Asp His Asp Leu Asp Phe Thr Pro
Glu Pro Ala Ala Arg 260 265 270 Ala Ser Phe Val Thr Gly Val Asn Cys
Glu Thr Ser Cys Val Gln Gln 275 280 285 Pro Pro Cys Phe Pro Leu Lys
Ile Pro Pro Asn Asp Pro Arg Ile Lys 290 295 300 Asn Gln Ala Asp Cys
Ile Pro Phe Phe Arg Ser Cys Pro Ala Cys Pro 305 310 315 320 Gly Ser
Asn Ile Thr Ile Arg Asn Gln Ile Asn Ala Leu Thr Ser Phe 325 330 335
Val Asp Ala Ser Met Val Tyr Gly Ser Glu Glu Pro Leu Ala Arg Asn 340
345 350 Leu Arg Asn Met Ser Asn Gln Leu Gly Leu Leu Ala Val Asn Gln
Arg 355 360 365 Phe Gln Asp Asn Gly Arg Ala Leu Leu Pro Phe Asp Asn
Leu His Asp 370 375 380 Asp Pro Cys Leu Leu Thr Asn Arg Ser Ala Arg
Ile Pro Cys Phe Leu 385 390 395 400 Ala Gly Asp Thr Arg Ser Ser Glu
Met Pro Glu Leu Thr Ser Met His 405 410 415 Thr Leu Leu Leu Arg Glu
His Asn Arg Leu Ala Thr Glu Leu Lys Ser 420 425 430 Leu Asn Pro Arg
Trp Asp Gly Glu Arg Leu Tyr Gln Glu Ala Arg Lys 435 440 445 Ile Val
Gly Ala Met Val Gln Ile Ile Thr Tyr Arg Asp Tyr Leu Pro 450 455 460
Leu Val Leu Gly Pro Thr Ala Met Arg Lys Tyr Leu Pro Thr Tyr Arg 465
470 475 480 Ser Tyr Asn Asp Ser Val Asp Pro Arg Ile Ala Asn Val Phe
Thr Asn 485 490 495 Ala Phe Arg Tyr Gly His Thr Leu Ile Gln Pro Phe
Met Phe Arg Leu 500 505 510 Asp Asn Arg Tyr Gln Pro Met Glu Pro Asn
Pro Arg Val Pro Leu Ser 515 520 525 Arg Val Phe Phe Ala Ser Trp Arg
Val Val Leu Glu Gly Gly Ile Asp 530 535 540 Pro Ile Leu Arg Gly Leu
Met Ala Thr Pro Ala Lys Leu Asn Arg Gln 545 550 555 560 Asn Gln Ile
Ala Val Asp Glu Ile Arg Glu Arg Leu Phe Glu Gln Val 565 570 575 Met
Arg Ile Gly Leu Asp Leu Pro Ala Leu Asn Met Gln Arg Ser Arg 580 585
590 Asp His Gly Leu Pro Gly Tyr Asn Ala Trp Arg Arg Phe Cys Gly Leu
595 600 605 Pro Gln Pro Glu Thr Val Gly Gln Leu Gly Thr Val Leu Arg
Asn Leu 610 615 620 Lys Leu Ala Arg Lys Leu Met Glu Gln Tyr Gly Thr
Pro Asn Asn Ile 625 630 635 640 Asp Ile Trp Met Gly Gly Val Ser Glu
Pro Leu Lys Arg Lys Gly Arg 645 650 655 Val Gly Pro Leu Leu Ala Cys
Ile Ile Gly Thr Gln Phe Arg Lys Leu 660 665 670 Arg Asp Gly Asp Arg
Phe Trp Trp Glu Asn Glu Gly Val Phe Ser Met 675 680 685 Gln Gln Arg
Gln Ala Leu Ala Gln Ile Ser Leu Pro Arg Ile Ile Cys 690 695 700 Asp
Asn Thr Gly Ile Thr Thr Val Ser Lys Asn Asn Ile Phe Met Ser 705 710
715 720 Asn Ser Tyr Pro Arg Asp Phe Val Asn Cys Ser Thr Leu Pro Ala
Leu 725 730 735 Asn Leu Ala Ser Trp Arg Glu Ala Ser 740 745
3720PRTHomo sapiens 3Met Arg Leu Leu Leu Gly Leu Ala Gly Leu Leu
Ala Val Leu Ile Met 1 5 10 15 Leu Gln Pro Ser Glu Gly Val Pro Pro
Ala Val Pro Gly Glu Val Asp 20 25 30 Thr Ser Val Val Leu Thr Cys
Met Glu Glu Ala Lys Arg Leu Val Asp 35 40 45 Lys Val Tyr Lys Glu
Arg Arg Glu Ser Ile Lys Gln Arg Leu His Ser 50 55 60 Gly Leu Ala
Ser Pro Met Glu Leu Leu Ser Tyr Phe Lys Gln Pro Val 65 70 75 80 Ala
Ala Thr Arg Thr Ala Val Arg Ala Ala Asp Tyr Leu His Val Ala 85 90
95 Leu Ser Leu Leu Glu Arg Lys Leu Arg Ala Leu Trp Pro Gly Arg Phe
100 105 110 Asn Val Thr Asp Val Leu Thr Pro Ala Gln Leu Asn Leu Leu
Ser Lys 115 120 125 Thr Ser Gly Cys Ala His Gln Asp Leu Gly Val Ser
Cys Pro Glu Lys 130 135 140 Asp Glu Tyr Arg Thr Ile Thr Gly Gln Cys
Asn Asn Arg Arg Ser Pro 145 150 155 160 Thr Leu Gly Ala Ser Asn Arg
Pro Phe Val Arg Trp Leu Pro Ala Glu 165 170 175 Tyr Glu Asp Gly Phe
Ser Leu Pro Phe Gly Trp Thr Pro Arg Val Lys 180 185 190 Arg Asn Gly
Phe Pro Val Pro Leu Ala Arg Ala Val Ser Asn Glu Ile 195 200 205 Val
Arg Phe Pro Thr Glu Lys Leu Thr Pro Asp Gln Gln Arg Ser Leu 210 215
220 Met Phe Met Gln Trp Gly Gln Leu Leu Asp His Asp Leu Asp Phe Ser
225 230 235 240 Pro Glu Pro Ala Ala Arg Val Ser Phe Leu Thr Gly Ile
Asn Cys Glu 245 250 255 Thr Ser Cys Leu Gln Gln Pro Pro Cys Phe Pro
Leu Lys Ile Pro Pro 260 265 270 Asn Asp Pro Arg Ile Lys Asn Gln Gln
Asp Cys Ile Pro Phe Phe Arg 275 280 285 Ser Ser Pro Ala Cys Thr Gln
Ser Asn Ile Thr Ile Arg Asn Gln Ile 290 295 300 Asn Ala Leu Thr Ser
Phe Val Asp Ala Ser Met Val Tyr Gly Ser Glu 305 310 315 320 Asp Pro
Leu Ala Met Arg Leu Arg Asn Leu Thr Asn Gln Leu Gly Leu 325 330 335
Leu Ala Val Asn Thr Arg Phe Gln Asp Asn Gly Arg Ala Leu Leu Pro 340
345 350 Phe Asp Thr Leu Arg His Asp Pro Cys Arg Leu Thr Asn Arg Ser
Ala 355 360 365 Asn Ile Pro Cys Phe Leu Ala Gly Asp Ser Arg Ala Ser
Glu Met Pro 370 375 380 Glu Leu Thr Ser Met His Thr Leu Phe Val Arg
Glu His Asn Arg Leu 385 390 395 400 Ala Lys Glu Leu Lys Arg Leu Asn
Ile Ala His Trp Asn Gly Glu Arg 405 410 415 Leu Tyr Gln Glu Ala Arg
Lys Ile Val Gly Ala Met Val Gln Ile Ile 420 425 430 Thr Tyr Arg Asp
Tyr Leu Pro Leu Val Leu Gly Arg Glu Ala Met Arg 435 440 445 Lys Tyr
Leu Arg Pro Tyr Cys Ser Tyr Asn Asp Ser Val Asp Pro Arg 450 455 460
Ile Ser Asn Val Phe Thr Asn Ala Phe Arg Tyr Gly His Thr Leu Ile 465
470 475 480 Gln Pro Phe Met Phe Arg Leu Asn Ser Arg Tyr Gln Pro Met
Gln Pro 485 490 495 Asn Pro Arg Val Pro Leu Ser Arg Val Phe Phe Ala
Ser Trp Arg Val 500 505 510 Val Leu Glu Gly Gly Ile Asp Pro Ile Leu
Arg Gly Leu Met Ala Thr 515 520 525 Pro Ala Lys Leu Asn Arg Gln Asn
Gln Ile Ala Val Asp Glu Ile Arg 530 535 540 Glu Arg Leu Phe Glu Gln
Val Met Arg Ile Gly Leu Asp Leu Pro Ala 545 550 555 560 Leu Asn Met
Gln Arg Ser Arg Asp His Gly Leu Pro Gly Tyr Asn Ala 565 570 575 Trp
Arg Arg Phe Cys Gly Leu Pro Val Pro Asn Thr Val Gly Glu Leu 580 585
590 Gly Thr Val Leu Arg Asn Leu Asp Leu Ala Arg Arg Leu Met Lys Leu
595 600 605 Tyr Gln Thr Pro Asn Asn Ile Asp Ile Trp Ile Gly Gly Val
Ala Glu 610 615 620 Pro Leu Asn Lys Asn Gly Arg Val Gly Pro Leu Leu
Ala Cys Leu Ile 625 630 635 640 Gly Thr Gln Phe Arg Lys Leu Arg Asp
Gly Asp Arg Phe Trp Trp Gln 645 650 655 Asn Lys Gly Val Phe Ser Lys
Lys Gln Gln Gln Ala Leu Ala Lys Ile 660 665 670 Ser Leu Pro Arg Ile
Ile Cys Asp Asn Thr Gly Ile Thr Phe Val Ser 675 680 685 Lys Asn Asn
Ile Phe Met Ser Asn Arg Phe Pro Arg Asp Phe Val Arg 690 695 700 Cys
Ser Arg Val Pro Ala Leu Asn Leu Ala Pro Trp Arg Glu Arg Arg 705 710
715 720
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